Plant Proteomics - Home - Springer978-3-540-72617-3/1.pdf · Plant Proteomics With 32 Figures, ... Zhentian Lei, Satish Nagaraj, ... 157 11.2 Two-Dimensional Electrophoretic Analysis

Jozef Šamaj ● Jay J. Thelen (Editors)

Plant Proteomics

Jozef ŠamajJay J. Thelen (Editors)

Plant Proteomics

With 32 Figures, 7 in color and 13 Tables

Dr. Jozef Šamaj Dr. Jay J. ThelenInstitute of Cellular and Molecular Botany University of Missouri-ColumbiaRheinische Friedrich-Wilhelms-University Bonn Department of BiochemistryDepartment of Plant Cell Biology Columbia, MO 65211Kirschallee 1 USAD-53115 BonnGermany

Institute of Plant Genetics and BiotechnologySlovak Academy of SciencesAkademicka 295007 NitraSlovak Republic

Library of Congress Control Number: 2007929273

ISBN-13 978-3-540-72616-6 Springer-Verlag Berlin Heidelberg New York

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Editors

Jozef Šamaj received his Ph.D. degree in Plant Physiology from the Comenius Uni-versity in Bratislava, Slovakia. He completed three post-doctoral programmes sup-ported by Eurosilva, the Alexander von Humboldt Foundation, and the EU Marie Curie Programme in the highly regarded laboratories of Alain Boudet in Toulouse, Dieter Volkmann in Bonn, and Heribert Hirt in Vienna. He worked on the cell biolo-gy of somatic embryogenesis, lignifi cation in tree species, arabinogalactan proteins, the cytoskeleton, and signalling proteins. Jozef Šamaj has co-edited three books and co-authored more than 75 research papers, reviews, and book chapters. He is a senior lecturer and group leader at the Institute of Cellular and Molecular Botany in Bonn, Germany, and senior researcher at the Institute of Plant Genetics and Bio-technology, Slovak Academy of Sciences, Nitra, Slovakia. His current research is focussed on the role of signalling components and the cytoskeleton in relation to the vesicular traffi cking during plant development and stress responses using integrated cell-biological and functional proteomics approaches.

Jozef Šamaj Jay Thelen

v

Jay Thelen received his B.Sc. degree in Biology and Biochemistry from the University of Nebraska-Lincoln in 1993. He earned his Ph.D. from the University of Missouri-Columbia (UMC) studying the structure and regulation of plant mitochondrial pyruvate dehydrogenase complexes under the guidance of Douglas Randall. In 1999 he started a 3-year postdoctoral position in John Ohlrogge’s lab at Michigan State University investigating the plastid acetyl-CoA carboxylase protein complex. He returned to UMC in 2002 as the Associate Director of a campus Proteomics Center. In 2004, he was promoted to Assistant Professor in the Biochemistry Department, a position he currently holds. He has authored or co-authored 35 research and review articles since 1994. His research interests are centered around the regulation of plant metabolism, particularly carbon assimilation in oilseeds, and multienzyme metabolic complexes. He is currently studying seed fi lling in numerous crop oilseeds, using various quantitative proteomics approaches. He is also investigating global phosphoprotein networks involved in seed development and is developing improved strategies for quantitative proteomics.

vi Editors

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

1. Introduction to Proteomics: a Brief Historical Perspective on Contemporary Approaches . . . . . . . . . . . . . . . . . . . . . . 1Jay J. Thelen

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Protein Separation and Detection for Proteome Investigations . . . . . 2

1.2.1 Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2.2 Two-Dimensional Gel Electrophoresis . . . . . . . . . . . . . . . . . 31.2.3 Extracting Proteins From Plant Samples. . . . . . . . . . . . . . . . 31.2.4 In-Gel Detection of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Protein Identification using Mass Spectrometry . . . . . . . . . . . . . . . . 51.3.1 Peptide Mass Fingerprint Protein Identification . . . . . . . . . . 61.3.2 Tandem Mass Spectrometry Protein

Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.4 Differential Protein Profiling Approaches . . . . . . . . . . . . . . . . . . . . . 8

1.4.1 Difference Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . 81.4.2 Chemical Labeling Approaches

for Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . 91.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2. High-Resolution Two-Dimensional Gel Electrophoresis: A Cornerstone of Plant Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14William J. Hurkman and Charlene K. Tanaka

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.2 Protein Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.2.1 Total Proteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2.2 Subproteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

vii

2.3 Protein Solubilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.4 Protein Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.5 Comparative Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3. An Introduction to Proteomics Data Analysis . . . . . . . . . . . . . . . . . . . . 29Curtis G. Wilkerson

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.2 Search Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.3 X!-Tandem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.4 Informatics Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.5 Relation Database Management of Large Scale Proteomics Data. . . 39References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4. Phosphoproteomics in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Sergio de la Fuente van Bentem, Thomas S. Nühse, and Heribert Hirt

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.2 Mitogen-Activated Protein Kinase Modules are Central

Components of Plant Signalling Pathways . . . . . . . . . . . . . . . . . . . . 424.3 Mass Spectrometry-Based Approaches to Identify

Phosphorylation Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.3.1 Enrichment Strategies for MS-based

Phosphoproteomic Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . 454.3.2 Global Identification of in vivo Phosphorylation

Sites using IMAC Coupled to MS Technology . . . . . . . . . . . . 464.4 Technologies Applicable to Studying Changes

in the Phosphoproteome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474.5 Plant Phosphoproteomics in the Future . . . . . . . . . . . . . . . . . . . . . . . 48References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

5. High-Throughput Identification of Plant Protein Kinase Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Birgit Kersten and Tanja Feilner

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535.2 Protein Microarray Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

5.2.1 History of Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555.2.2 Types of Protein Microarrays. . . . . . . . . . . . . . . . . . . . . . . . . 575.2.3 Establishment of Protein Microarrays

for Phosphorylation Studies. . . . . . . . . . . . . . . . . . . . . . . . . . 575.3 Using Plant Protein Microarrays for the Identification

of Potential Protein Kinase Substrates. . . . . . . . . . . . . . . . . . . . . . . . 595.4 Potential Substrates of Arabidopsis MPK3 and MPK6 . . . . . . . . . . . 62

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5.4.1 Brief Bibliographic Overview of MAPK Pathway . . . . . . . . . 625.4.2 Large-Scale Identification of Potential MPK3

and MPK6 Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645.5 Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . 65References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6. Discovery via Proteomics of a Novel Cell Signalling Pathway in Plants Involving Extracellular ATP . . . . . . . . . . . . . . . . . . 71Stephen Chivasa, William J. Simon, John M. Hamilton, Keith Lindsey, and Antoni R. Slabas

6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726.2 Proteomics of the ECM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

6.2.1 The Plant ECM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726.2.2 Extraction of Plant ECM Proteins . . . . . . . . . . . . . . . . . . . . . 736.2.3 Identity of ECM Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

6.3 Unravelling New Biological Functions of the Apoplast . . . . . . . . . . 756.3.1 Phosphorylated and ATP-Binding ECM Proteins . . . . . . . . . 756.3.2 Plant Extracellular ATP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776.3.3 In Search of the Role of Extracellular ATP . . . . . . . . . . . . . . 78

6.4 Physiological Significance of Extracellular ATP Depletion-Induced Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796.4.1 Elicitor-Induced Cell Death is Regulated

by External ATP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806.5 How Does Extracellular ATP Function? . . . . . . . . . . . . . . . . . . . . . . 816.6 Conclusions and Future Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

7. Cereal Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Hisashi Hirano

7.1 Introduction: Genomic and Proteomic Analyses . . . . . . . . . . . . . . . . 877.2 A Typical Procedure for Proteomic Analysis . . . . . . . . . . . . . . . . . . 897.3 Mass Spectrometry and Proteomic Analysis . . . . . . . . . . . . . . . . . . . 897.4 Expression Profiles of Cereal Proteins. . . . . . . . . . . . . . . . . . . . . . . . 90

7.4.1 2-DE-Based Profiling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907.4.2 Shotgun Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927.4.3 Quantitative Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937.4.4 Subcellular Localization of Cereal Proteins . . . . . . . . . . . . . 94

7.5 Post-Translational Modifications of Cereal Proteins . . . . . . . . . . . . . 947.5.1 Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947.5.2 Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957.5.3 Other Post-Translational Modifications . . . . . . . . . . . . . . . . 97

7.6 Protein-Protein Interactions in Cereals . . . . . . . . . . . . . . . . . . . . . . . 977.6.1 Immunoaffinity Purification/MS of Protein Complexes . . . . . 987.6.2 Tandem Affinity Purification/MS of Protein Complexes . . . . 98

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7.6.3 Blue Native-/SDS-PAGE/MS of Protein Complexes. . . . . . 987.6.4 Protein Interaction Analysis Using

Chromosome-Deletion Lines . . . . . . . . . . . . . . . . . . . . . . . 99 7.7 Proteome Informatics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 7.8 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

8. Current Status of Arabidopsis Thaliana Proteomics . . . . . . . . . . . . . . 105Sacha Baginsky and Wilhelm Gruissem

8.1 Current Status of Arabidopsis Proteomics. . . . . . . . . . . . . . . . . . . 105 8.2 Analysis of Post-Translational Modifications . . . . . . . . . . . . . . . . 108 8.3 Establishing Protein/Protein Interactions. . . . . . . . . . . . . . . . . . . . 110 8.4 Revealing Proteome Dynamics by Relative

and Absolute Protein Quantification . . . . . . . . . . . . . . . . . . . . . . . 111 8.5 Towards a Complete Proteome Map for Arabidopsis . . . . . . . . . . 113 8.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

9. Proteomics of Medicago truncatula . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Zhentian Lei, Satish Nagaraj, Bonnie S. Watson, and Lloyd W. Sumner

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 9.2 Proteomics: an Important Tool in Dissecting

Legume Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 9.3 Proteomics of M. truncatula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

9.3.1 Proteomics of M. truncatula: Microbe Interactions . . . . . . 1269.3.2 Proteomics of M. truncatula: Tissues . . . . . . . . . . . . . . . . . 129

9.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

10. Proteomics of Seed Development in Oilseed Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Martin Hajduch, Ganesh Kumar Agrawal, and Jay J. Thelen

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13810.2 Two-Dimensional Electrophoresis to Profile Protein

Expression During Seed Filling in Soybean . . . . . . . . . . . . . . . . . 13910.3 Proteomic Analysis of Seed Filling in Rapeseed. . . . . . . . . . . . . . 14210.4 Comparison of Carbon Assimilation During Seed

Filling in Soybean and Rapeseed . . . . . . . . . . . . . . . . . . . . . . . . . . 14310.5 Proteomics of Mature Soybean Seed . . . . . . . . . . . . . . . . . . . . . . . 14610.6 Organelle and Tissue Specific Proteomics – Towards

“In-Depth” Oilseed Proteome Characterization. . . . . . . . . . . . . . . 148

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10.7 Presence of Functionally Diverse Phosphoproteins in Developing Rapeseed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

10.8 Conclusions and Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . 151References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

11. Proteome Analysis of Nicotiana tabacum Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155Kris Laukens, Noor Remmerie, Thomas De Vijlder, Kim Henderickx, and Erwin Witters

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15511.1.1 The Tobacco BY-2 Cell Suspension . . . . . . . . . . . . . . . . . 15611.1.2 Analytical Work Flow Considerations . . . . . . . . . . . . . . . 157

11.2 Two-Dimensional Electrophoretic Analysis of Tobacco Cell Suspension Proteomes . . . . . . . . . . . . . . . . . . . . . 15811.2.1 Tobacco BY-2 Proteome: a Reference Map . . . . . . . . . . . 15911.2.2 The Tobacco Phosphoproteome: Effects

of Bacterial Lipopolysaccharides . . . . . . . . . . . . . . . . . . 16011.3 Proteome Analysis of Tobacco BY-2 Plastids . . . . . . . . . . . . . . . . 16111.4 Blue-Native/SDS PAGE Analysis of Tobacco

BY-2 Protein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16211.5 Identification of Tobacco Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 16411.6 Data Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

12. Cell Wall Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169Georges Boudart, Zoran Minic, Cécile Albenne, Hervé Canut, Elisabeth Jamet, and Rafael F. Pont-Lezica

12.1 Introduction: Cell Wall Proteins Before Proteomics . . . . . . . . . . . 17012.2 The Surprising Variety of Cell Wall Proteins. . . . . . . . . . . . . . . . . 170

12.2.1 Glycoside Hydrolases. . . . . . . . . . . . . . . . . . . . . . . . . . . . 17212.2.2 Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17512.2.3 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17612.2.4 Protein Inhibitors of Cell Wall Modifying

Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17612.3 Proteomics as an Efficient Way to Depict Cell Wall

Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17712.3.1 N-Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17712.3.2 Proline Hydroxylation and O-Glycosylation . . . . . . . . . . 17812.3.3 Addition of GPI Anchors . . . . . . . . . . . . . . . . . . . . . . . . . 17912.3.4 Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

12.4 Emerging Functions for Cell Wall Proteins . . . . . . . . . . . . . . . . . . 18012.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

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13. Plasma Membrane Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186Erik Alexandersson, Niklas Gustavsson, Katja Bernfur, Per Kjellbom, and Christer Larsson

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18613.2 Plasma Membrane Purification and Fractionation. . . . . . . . . . . . . 188

13.2.1 Plant Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18813.2.2 Cell Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18813.2.3 Washing and Extraction Methods . . . . . . . . . . . . . . . . . . 18913.2.4 Assessment of Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

13.3 Protein Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19313.3.1 Gel-Based Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 19313.3.2 Non-Gel-Based Techniques . . . . . . . . . . . . . . . . . . . . . . . 194

13.4 Protein Digestion for MS-Analysis . . . . . . . . . . . . . . . . . . . . . . . . 19413.5 Biological Insights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

13.5.1 Proteins Identified in the Plasma Membrane. . . . . . . . . . 19513.5.2 Studies Targeted at Plasma Membrane Proteins

with Specific Properties . . . . . . . . . . . . . . . . . . . . . . . . . . 19713.5.3 Differential Plasma Membrane Proteomics. . . . . . . . . . . 19713.5.4 Overlap of Identified Proteins Among Studies. . . . . . . . . 19813.5.5 Contaminating Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 200

13.6 Conclusions and Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . 201References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

14. The Proteomes of Chloroplasts and other Plastids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Paul Jarvis

14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20714.2 Proteome Catalogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

14.2.1 The Thylakoid Membrane System . . . . . . . . . . . . . . . . . . 20914.2.2 The Envelope Membrane System . . . . . . . . . . . . . . . . . . . 21114.2.3 The Stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21214.2.4 Plastoglobules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21414.2.5 Whole Organelles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

14.3 Protein Targeting Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21614.4 Comparative Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21914.5 Multiprotein Complexes and Protein Modification . . . . . . . . . . . . 21914.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

15. The Plant Mitochondrial Proteome. . . . . . . . . . . . . . . . . . . . . . . . . . . . 226A. Harvey Millar

15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22615.2 Isolation of Plant Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

xii Contents

15.3 Experimental Analysis of the Proteome and Future Plans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

15.4 Advances in Predicting the Remaining Proteome . . . . . . . . . . . . 234 15.5 Beyond the List of the Proteome to its Regulation

and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 15.5.1 Post-Translational Modifications. . . . . . . . . . . . . . . . . . 236 15.5.2 Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 15.5.3 Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

15.6 Non-Covalent Interactions Within the Proteome. . . . . . . . . . . . . 239 15.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

16. Proteomic Analysis of the Plant Nucleolus . . . . . . . . . . . . . . . . . . . . . . 247Olga Koroleva, Peter McKeown, Alison Pendle, and Peter Shaw

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 16.2 Proteomics Techniques used for Plants . . . . . . . . . . . . . . . . . . . . 249 16.3 Purification of Nucleoli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 16.4 Isolation and Analysis of Arabidopsis Nucleolar

Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 16.5 Analysis of Post-Translational Protein Modifications . . . . . . . . . 253 16.6 Quantification Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

16.6.1 In Vivo Isotope Labelling: SILAC . . . . . . . . . . . . . . . . . 256 16.6.2 In Vitro Isotope Labelling . . . . . . . . . . . . . . . . . . . . . . . 257 16.6.3 Comparison of Quantification Methods. . . . . . . . . . . . . 258

16.7 Proteomic Analysis of Nuclear Protein Complexes. . . . . . . . . . . 258 16.7.1 TAP Tag Strategy and Variations . . . . . . . . . . . . . . . . . . 258 16.7.2 TAP Tag Purification of Nuclear Transport

Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 16.8 Reliability of Proteomic Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 16.9 Data Analysis and Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26316.10 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265Refernces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

17. Pollen and Pollen Tube Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270Tong Chen, Xiaoqin Wu, Yanmei Chen, Nils Böhm, Jinxing Lin, and Jozef Šamaj

17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 17.2 Procedure for Pollen Proteomic Analysis . . . . . . . . . . . . . . . . . . 272 17.3 Proteomics Focused on Pollen Development. . . . . . . . . . . . . . . . 273

17.3.1 Angiosperm Pollen Proteomes . . . . . . . . . . . . . . . . . . . . 273 17.3.2 Proteomic Analysis of Gymnosperm Pollen

and Pollen Tube Development . . . . . . . . . . . . . . . . . . . . . 277 17.4 Conclusions and Future Perspectives. . . . . . . . . . . . . . . . . . . . . . 279References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Contents xiii

18. Plant Proteomics Upon Fungal Attack . . . . . . . . . . . . . . . . . . . . . . . . . 283Frank Colditz, Franziska Krajinski, and Karsten Niehaus

18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28318.2 Types of Plant–Fungi Interactions . . . . . . . . . . . . . . . . . . . . . . . . . 285

18.2.1 Mutualistic Plant–Fungi Interactions with Microsymbionts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

18.2.2 Parasitic Plant–Fungi Interactions – Infection Strategies of Fungal Pathogens . . . . . . . . . . . . . . . . . . . . 286

18.2.3 Model Plant/Fungi Pathosystems. . . . . . . . . . . . . . . . . . . 28818.3 The “Fungal Side” – Proteomics of Fungal Pathogens . . . . . . . . . 289

18.3.1 Fungal and Oomycete Effector Proteins . . . . . . . . . . . . . 28918.3.2 Pathogen-Associated Molecular Patterns . . . . . . . . . . . . 292

18.4 The “Plant Side” – Plant Proteomics Upon Fungal Attack . . . . . . 29318.4.1 Inducible Defence-Related Proteins and their

Occurrence in Infected Plants . . . . . . . . . . . . . . . . . . . . . 29418.4.2 Induction of Systemic Acquired Resistance

by Fungal Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29718.4.3 Mapping of Protein Phosphorylation Patterns

Triggered by Fungal Pathogens or Elicitors . . . . . . . . . . 29818.5 Induction of Defence-Related Proteins in Non-Pathogenic

Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30018.6 Conclusions and Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . 300References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

19. Metabolic Intricacies of the Symbiotic Association between Soybean and Bradyrhizobium japonicum: A Proteomic Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310Annamraju D. Sarma, Nathan W. Oehrle, and David W. Emerich

19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31019.2 Symbiotic Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31119.3 Mitochondrial Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31319.4 Nodule Cytosol Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31319.5 Symbiosome Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31819.6 Bacteroid Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31919.7 An Integrated Proteomic View of the Soybean Nodule . . . . . . . . . 321References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

20. Proteomes in Arbuscular Mycorrhizal Symbiosis . . . . . . . . . . . . . . . . 326Eliane Dumas-Gaudot and Ghislaine Recorbet

20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32620.2 Trends in AM Symbiosis Towards Simultaneous

“…omics” Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

xiv Contents

20.2.1 Combining Protein, Transcript and Metabolite Extractions for Investigation of AM Root/Fungus Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

20.2.2 Experimental Plant Design . . . . . . . . . . . . . . . . . . . . . . . 33120.3 Proteome Profiling During AM Symbiosis . . . . . . . . . . . . . . . . . . 331

20.3.1 Proteomics of Total Proteins . . . . . . . . . . . . . . . . . . . . . . 33420.3.2 Sub-Cellular Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . 335

20.4 Further Insights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33720.5 Conclusions and Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . 338References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

21. Plant Proteome Responses to Abiotic Stress. . . . . . . . . . . . . . . . . . . . . 346Delphine Vincent and Michel Zivy

21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34721.2 Water Deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34721.3 Salt Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35121.4 Cold Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35421.5 Heat Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35621.6 High Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35721.7 Metal Stress and Herbicides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35821.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

Contents xv

Preface

Plant proteomics is a relatively new research fi eld focused on the large-scale functional analysis of proteins extracted from intact plants, particular plant organs, tissues, individual cells, subcellular organelles and/or separated suborganellar struc-tures. Rapidly increasing numbers of excellent publications on plant proteomics, both in primary and applied research, demonstrate the imense potential and impor-tance of this research fi eld for current and future plant science. One of the main aims of plant proteomics is to study the assembly and functional interactions of plant proteins. Proteins often function as molecular machines organized into multiprotein complexes localized within specialized subcellular compartments. Enormous meth-odological and technical developments have moved recent proteomics towards the large-scale study of post-translational modifi cations of proteins involved in cellular signalling (regulated by reversible phosphorylation), protein turnover (ubiquitinyla-tion) and membrane association (palmitoylation and myristylation).

This book highlights this rapid progress in plant proteomics with emphasis on model species, subcellular organelles as well as specific aspects such as signalling, plant reproduction, stress biology and/or pathogen/symbiotic interactions between plants and microorganisms. Additionally, brief historical overviews on plant pro-teomics and two-dimensional gel electrophoresis as well as an introduction to bio-infomatics are provided here. Thus, this monograph represents a synthesis of the most current knowledge in this field, including the most important biological aspects as well as new methodological approaches such as high-resolution two-dimensional electrophoresis, protein microchips, MudPIT (multidimensional pro-tein identification technology), fluorescent DIGE (difference gel electrophoresis) alone and/or in combination with stable isotope reagents such as ICAT (isotope-coded affinity tag) and iTRAQ (isobaric tag for relative and absolute quantitation), which allow relative protein quantification. The reader is provided with an up-to date view on plant proteomes in carefully selected model plant species such as Arabidopsis, cereals, legumes and oil seed plants. One chapter focuses on the cell division model represented by suspension cultured tobacco BY-2 cells. Several chapters are devoted to the proteomics of plant organelles and compartments. Among the latter, special attention is paid to the cell wall, plasma membrane, plas-tids, mitochondria and nucleolus. Two chapters focus on proteomic approaches used to study plant reproduction, namely pollen proteomics and the proteomics of

xvii

seed development in oilseed crops. Finally, four chapters describe proteomes dur-ing pathogenic and symbiotic interactions between plants and microrganisms, and during plant stress responses. Regarding future perspectives, it is very important that diverse integrated approaches including advanced proteomic techniques com-bined with functional genomics, bioinformatics, metabolomics and/or with advanced molecular cell biology are nicely presented in several chapters. Thus, this book not only covers the rapid progress in the field of plant proteomics but also delivers this recent knowledge to a broad spectrum of readers including advanced students, teachers and researchers.

At this point I would like to thank my co-editor Jay Thelen and all the authors for their great job and excellent contributions to this book. Last but not least, my special thanks goes to my family, my wife Olinka and sons Matejko and Tomáško, for their encouragement and patience with me during this book project.

Bonn, April 2007 Jozef Šamaj

xviii Preface

List of Abbreviations

Chapter 1PAGE polyacrylamide gel electrophoresisSDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresisLC liquid chromatography2-D two-dimensionalIPG immobilized pH gradientIEF isoelectric focusingCBB Coomassie Brilliant BlueESI Electrospray IonizationMALDI Matrix Assisted Laser Desorption IonizationTOF Time of FlightPMF peptide mass fi ngerprintingEST expressed sequence tagICAT Isotope-Coded Affi nity TagMS Mass spectrometry

Chapter 22-DE Two-dimensional gel electrophoresisMS mass spectrometryIPG immobilized pH gradientsNEPHGE non-equilibrium pH gradient electrophoresismABC1 mitochondrial ATP-binding cassette protein 1SB 3-10 N-decyl-N, N-dimethyl-3-ammonio-1-propane sulfonateTBP tributyl phosphineDIGE difference gel electrophoresisMALDI Matrix-assisted laser desorption/ionizationTOF Time of fl ightCID collision-inducedIEF isoelectric focusingSDS-PAGE sodium dodecyl sulfate polyacrylamide

xix

gel electrophoresisEST expressed sequence tagCBB Coomassie Brilliant Blue

Chapter 3CID collision-induced dissociationPTM post-translational modifi cationGPM global proteome machine

Chapter 4MAPK mitogen-activated protein kinaseMAPKKKs MAPK kinase kinasesMAPKKs MAPK kinasesPP2C Protein Ser/Thr phosphatase 2CDsPs Ser/Thr/Tyr phosphatasesPTP protein Tyr phosphataseMS mass spectrometryLC-MS liquid chromatography-mass spectrometryIMAC immobilised metal ion affi nity chromatographySCX strong cationic exchangeSRPK SR protein-specifi c kinaseSILAC Stable Isotope Labelling by Amino acids in Cell cultureTiO

2 Titanium dioxide

EST expressed sequence tag

Chapter 52-DE two-dimensional gel electrophoresisMS mass-spectrometryUPA universal protein arrayPMAs protein microarraysAMAs antibody microarraysRPMAs reverse protein microarraysHMG high mobility groupACS-6 1-aminocyclopropane-1-carboxylic acid synthase-6MKS1 MAPK substrate 1VSP1 vegetative storage protein1Pro-Q DPS ProQ-Diamond phosphoprotein strainCK2α casein kinase2αMPK mitogen-activated kinasePKA Protein kinase A

Chapter 6ECM extracellular matrixER endoplasmic reticulum

xx List of Abbreviations

GAPDH glyceraldehyde-3-phosphate dehydrogenase2D-DiGE 2-dimensional difference gel electrophoresis2-DE 2-dimensional gel electrophoresisMS mass-spectrometryGFP green fl uorescent proteinFB1 fumonisin B1

Chapter 72-DE 2-dimensional gel electrophoresisBN-PAGE blue native-polyacrylamide gel electrophoresisCK2α casein kinase 2αDIGE difference gel electrophoresisDLC diamond-like carbon coated stainless steelESI electrospray ionizationFT Fourier transform ion cyclotron resonanceGFP green fl uorescent proteinICAT isotope-coded affi nity tagIMAC immobilized metal affi nity chromatographyIT ion trapiTRAQ isobaric tag for relative and absolute quantitationLC liquid chromatographyMALDI matrix-assisted laser desorption ionizationMS mass spectrometrySDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresisPTM post-translational modifi cationQ quadrupoleTAP tandem affi nity purifi cationTOF time-of-fl ight2-DE two-dimensional electrophoresis

Chapter 8MS mass-spectrometryMALDI matrix-assisted laser desorption ionizationQ quadrupoleTOF time-of-fl ightESI electrospray ionizationeSLDB eukaryotic sub-cellular localisation databaseSUBA subcellular location database for Arabidopsis proteinsGFP green fl uorescent proteinAMPDB Arabidopsis Mitochondrial Protein DatabaseIMAC metal ion affi nity chromatographyTRX thioredoxinTAP tandem affi nity purifi cation

List of Abbreviations xxi

TEV tobacco etch virusICAT isotope-coded affi nity tagiTRAQ isobaric tag for relative and absolute quantitationSILAC stable isotope labeling with amino acids in cell cultureMRM multiple reaction monitoringHPLC high performance liquid chromatographyLC liquid chromatography

Chapter 92-DE two-dimensional electrophoresisMb MegabaseEST expressed sequence tagTC tentative consensusppm parts per millionIEF isoelectric focusingAM arbuscular mycorrhizaldai days after inoculationPR pathogenesis-relatedABA abscisic acidAdo-Met S-adenosyl-MetLEA late embryogenesis abundantA. euteiches Aphanomyces euteichesNSF N-ethylmaleimide-sensitive fusionABA abscisic acidG. mosseae Glomus mosseaeG. intraradices Glomus intraradicesiTRAQ isobaric tags for relative and absolute quantitationICAT isotope-coded affi nity tagsIPG immobilized pH gradientLC-MS/MS liquid chromatography coupled to tandem mass spectrometryMALDI TOF-MS matrix assisted laser desorption/ionization time-of-fl ight mass spectrometryMS mass spectrometryMS/MS tandem mass spectrometryM. truncatula Medicago truncatulaMudPIT multidimensional protein identifi cation technologypI isoelectric pointPMF peptide mass fi ngerprintingPR pathogenesis-relatedRuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenaseS. meliloti Sinorhizobium meliloti.

Chapter 10WAF weeks after fl owering

xxii List of Abbreviations

2-DE two-dimensional gel electrophoresisPTM post-translational modifi cationsPMF peptide mass fi ngerprintTC tentative consensusIPG immobilized pH gradientMALDI-TOF Matrix Assisted Laser Desorption Ionization-Time of FlightEST expressed sequence tagSSP seed storage proteinsLOX lipoxygenasesSuSy sucrose synthaseSBP sucrose-binding proteinPEP phosphoenolpyruvatePDC pyruvate dehydrogenase complexFBA fructose bisphosphate aldolaseRuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase 3-PGA 3-phosphoglycerateGAPDH glyceraldehyde-3-phosphate dehydrogenasePGK phosphoglycerate kinasePGM phosphoglucomutasePGI phosphoglucose isomeraseTPI triose-phosphate isomerasesiPGAM 2,3-bisphosphoglycerate-independent phopsphoglycerate mutasePK pyruvate kinaseLEA late embryogenesisIEF isoelectric focusingTAG triacylglycerolER endoplasmic reticulumBiP luminal binding proteinMS/MS tandem mass spectrometryCBB Coomassie Brilliant Blue

Chapter 11EST expressed sequence tagBY-2 Bright Yellow-22-DE two dimensional gel-electrophoresisPTM post-translational modifi cationMS mass spectroscopyPMF peptide mass fi ngerprintDiGE difference in gel electrophoresisiTRAQ isobaric tags for relative and absolute quantifi cationLC liquid chromatographyIPG immobilised pH gradientSDS-PAGE sodium dodecyl sulfate polyacrylamide

List of Abbreviations xxiii

gel electrophoresisIEF isoelectric focusingLC-ESI-Q-TOF MS liquid chromatography–electrospray ionisation–quadrupole–time-of-fl ight mass spectrometryMALDI matrix-assisted laser desorption/ionizationMS/MS tandem MSLPS lipopolysaccharidesRuBP ruthenium II tris bathophenanthroline disulfonateROS reactive oxygen speciesBN blue nativeRuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase

Chapter 12CWP cell wall proteinHRGPs Hyp-rich glycoproteinsH/PRPs Hyp/Pro-rich proteinsGRPs Gly-rich proteinsGH glycoside hydrolasesXTH xyloglucan endotransglucosylase/hydrolasesPMEs pectin methylesterasesLRX leucine-rich repeat-extensinsAGPs arabinogalactan proteinsMS mass spectrometryPTM post-translational modifi cationsCWME cell wall modifying enzymesCWMEI inhibitors of cell wall modifying enzymesPG polygalaturonaseXEGIPs xyloglucan endoglucanase inhibiting proteinsGPI glycosylphosphatidylinositolMALDI-TOF Matrix-assisted laser desorption/ionization-time of fl ightTDIF tracheary differentiation inhibitory factorPGIPs inhibitors of polygalacturonasesPMEI Inhibitor of PMENt-CIF inhibitor of tobacco invertaseER endoplasmic reticulum

Chapter 13MS mass spectroscopyRLKs receptor-like protein kinasesIEF iso-electrofocusingCTAB cationic trimethyl ammonium bromideBN-PAGE blue-native electrophoresisMuDPIT multidimensional protein identifi cation technique

xxiv List of Abbreviations

MALDI matrix assisted laser desorption/ionisationESI electrospray ionisationPI-PLC phosphatidylinositol phospholipase CIMAC immobilised metal ion affi nity chromatographyLDS lithium dodecyl sulphateLOPIT localisation of organelle proteins by isotope taggingGPI glycosylphosphatidylinositoliTRAQ isobaric tags for relative and absolute quantifi cationER endoplasmic reticulum2-DE two-dimensional gel electrophoresis16-BAC benzyldimethyl-n-hexadecylammonium chlorideCNBr cyanogen bromideGO gene ontologyDIGE difference in gel electrophoresisESTs expressed sequence tags

Chapter 14MS mass spectroscopyPTM post-translational modifi cationTIC Translocon at the Inner envelope membrane of ChloroplastsTOC Translocon at the Outer envelope membrane of Chloroplasts2-DE two-dimensional gel electrophoresisTAT twin-arginine translocationTPR tetratricopeptidePPR pentatricopeptidePAP plastid lipid-associated proteinER endoplasmic reticulumceQORH chloroplast envelope quinone oxidoreductase homologueIEP32 inner envelope protein of 32 kDaCAH1 carbonic anhydrase 1Clp caseinolytic proteaseIEF isoelectric focusing

Chapter 15BSA bovine serum albuminPVP polyvinylpyrrolidoneFW fresh weightPAGE Polyacrylamide gel electrophoresisIEF isoelectric focusingSDS-PAGE sodium dodecyl sulfate PAGE

List of Abbreviations xxv

MS mass spectrometryBN blue nativeTOM translocase of the outer membraneHSPs heat shock proteinsCMS cytoplasmic male sterilityPTM post-translational modifi cationPDC pyruvate dehydrogenase complexTCA cycle tricarboxylic acid cycleAOS active oxygen speciesROS reactive oxygen speciesHNE 4-hydroxy-2-nonenalTRX thioredoxinY2H yeast two-hybrid techniqueFRET fl uorescence resonance energy transferBRET bioluminescence resonance energy transferGRAVY grand average of hydrophobicity

Chapter 16NoLS Nucleolar localisation sequencesPLRV potato leaf-roll virusGFP green fl uorescent protein2-DE 2D polyacrylamide gel electrophoresisLC liquid chromatographyMS mass spectrometryMr relative molecular massDiGE difference gel electrophoresisMuDPIT multidimensional protein identifi cation techniqueMALDI matrix assisted laser desorption/ionisationESI electrospray ionisationMS/MS tandem MSTOF time-of-fl ightFT-ICR-MS Fourier-transform ion-cyclotron resonance mass spectrometersnRNP small nuclear RNPPTMs Post-translational modifi cationsGPI glycosylphosphatidylinositolSILAC Stable Isotope Labelling by Amino acids in Cell cultureICAT isotope coded affi nity taggingLOPIT Localisation of organelle proteins by isotope taggingER endoplasmic reticulumITRAQ Isobaric tag for relative and absolute quantifi cationTAP tandem affi nity purifi cationTEV tobacco etch-virusCBP calmodulin-binding protein

xxvi List of Abbreviations

EF elongation factorEST expressed sequence tag

Chapter 17PTMs Post-translational modifi cationsMS mass spectrometryMS/MS tandem mass spectrometry2-DE two-dimensional gel electrophoresisGO gene ontologyEST expressed sequence tagQ quadrupoleESI electrospray ionisationMALDI-TOF matrix assisted laser desorption/ionisation–time-of-fl ight

Chapter 18PAMPs pathogen-associated molecular patternsPRs pathogenesis-related proteinsSAR systemic acquired resistanceROS reactive oxygen speciesPR pathogenesis-relatedSA salicylic acidJA jasmonic acidET ethyleneMS mass spectrometryAM arbuscular mycorrhizal2D-LC two dimensional liquid chromatographyMS/MS tandem mass spectrometryNep1 necrosis- and ethylene-inducing peptideHR hypersensitive responseMAPKs mitogen-activated protein kinasesEST expressed sequence tagCSI cross-species identifi cationAVR avirulenceR resistanceRLP receptor-like proteinCITRX Cf-9-interacting thioredoxinNBS-LRR nucleotide binding site leucine-rich repeatGIP glucanase inhibitor proteinsPI protease inhibitorsTGases transglutaminasesCBEL cellulose binding elicitor lectinPAMP pathogen-associated molecular patternNLPs Nep1-like proteinsCBD cellulose-binding domain

List of Abbreviations xxvii

PRLs PR-like proteinsPAL phenylalanine ammonia-lyaseTMV tobacco mosaic virusFHB Fusarium head blightSOD superoxide dismutaseHRGP hydroxyproline-rich glycoproteinSAR systemic acquired resistance

Chapter 192-DE two-dimensional electrophoresisMALDI matrix assisted laser desorption ionisationPMF peptide mass fi ngerprintingCDPK calmodulin-like domain protein kinaseNDPK Nucleoside diphosphate kinase

Chapter 20AM arbuscular mycorrhizal1D-SDS PAGE one dimensional sodium dodecyl sulfate polycacrylamide gel2-DE two-dimensional gel electrophoresisLC liquid chromatographyESI-MS/MS electrospray ionisation tandem mass spectrometryESI-Q-TOF electrospray ionisation quadrupole time of fl ightESTs expressed sequence tagsGPI glycosylphosphatidylinositolHPLC high performance liquid chromatographyIPG immobilized pH gradientMALDI-TOF matrix assisted laser desorption ionisation time of fl ightPCR polymerase chain reactionPM plasma membraneRNAi RNA interferenceTILLING targeting induced local lesions in genomesPMF peptide mass fi ngerprintMS/MS tandem mass spectrometryHPLC high performance liquid chromatographyDIGE 2-D difference gel electrophoresisICAT isotope coded affi nity tagMUDPIT multidimensional protein identifi cation technologyRNAi RNA interference

Chapter 21MS Mass spectrometryMALDI-TOF matrix assisted laser desorption ionisation time of fl ight

xxviii List of Abbreviations

ABA Abscisic acidRuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenaseHSP heat shock protein2-DE two-dimensional electrophoresisPR pathogenesis-relatedASR ABA/stress/ripening responsive proteinCOMT caffeate-O-methyltransferaseSAM S-adenosyl-l-methionineQTLs quantitative trait lociPQL protein quantity locusLEA late embryogenesis abundantCV coeffi cient of variationROS reactive oxygen speciesDIGE Difference in-gel electrophoresisPMF peptide mass fi ngerprintEIFs eukaryotic initiation factorsSOD superoxide dismutasePG plastoglobulePC phytochelatinGSH glutathioneBN Blue nativeGST Glutathione S-transferase

List of Abbreviations xxix