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Proteome Changes Following Biotic and Abiotic Stress in Forest Trees

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Proteome Changes Following Biotic and Abiotic Stress in Forest Trees Technische Universität München Fachgebiet Forstgenetik Proteome changes following biotic and abiotic stress in forest trees Cristina-Maria Vâlcu Vollständiger Abdruck der von der Fakultät für Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. G. Wenzel Prüfer der Dissertation: 1. Univ.-Prof. Dr. G. Müller-Starck 2. apl. Prof. Dr. A. Görg 3. Univ.-Prof. Dr. A. Polle, Georg-August-Universität Göttingen (Schriftliche Beurteilung) Die Dissertation wurde am 04.10.2007 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 10.12.2007 angenommen. 2 TABLE OF CONTENTS Contribution to published papers 4 Index of figures and tables 5 List of abbreviations 6 1 INTRODUCTION 7 1.1 Plant stress in the context of climate change 7 1.2 Abiotic stress in forest trees: elevated temperature 8 1.3 Biotic stress in forest trees: pathogen and herbivore stress 9 1.4 Proteomics technologies for the study of stress response in plant species 11 1.5 Objectives of the present investigations 12 2 GENERAL METHODOLOGY 13 2.1 Optimisation of two dimensional gel electrophoresis separations 13 2.2 Experimental setup for the study of proteome changes following abiotic and biotic stress 16 2.2.1 Abiotic stress: Picea abies response to and recovery from heat stress 16 2.2.2 Biotic stress: Fagus sylvatica defence response to pathogen attack and wounding 17 3 RESULTS 19 3.1 Optimised two dimensional gel electrophoresis separations of woody plant proteins 19 3.2 Abiotic stress: Picea abies response to and recovery from heat stress 20 3.3 Biotic stress: Fagus sylvatica defence response to pathogen attack and wounding 21 4 DISCUSSIONS 22 4.1 Proteomics technologies for differential display experiments 22 4.2 Abiotic stress: Picea abies response to and recovery from heat stress 24 4.3 Biotic stress: Fagus sylvatica defence response to pathogen attack and wounding 26 5 CONCLUSIONS 29 6 SUMARY 30 7 REFERENCES 31 8 APPENDIXES: [1] Reduction of proteins during sample preparation and two-dimensional electrophoresis of woody plant samples. Vâlcu CM, Schlink K. [2] Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis. Vâlcu CM, Schlink K. [3] Reproducibility of two-dimensional electrophoresis at different replication levels, Vâlcu CM, Vâlcu M. [4] Protein expression during the recovery from heat stress of Norway spruce (Picea abies) provenances from contrasting elevations, Vâlcu CM, Lalanne C, Plomion C, Schlink K. [5] Protein polymorphism between two Picea abies populations revealed by two-dimensional electrophoresis and tandem mass spectrometry, Vâlcu CM, Lalanne C, Müller-Starck G, Plomion C, Schlink K. [6] Local and systemic changes in European beech (Fagus sylvatica) proteome following infection with Phytophthora citricola, Vâlcu CM, et al. 3 Contribution to published papers This dissertation is based on the following publications: 1 Vâlcu CM, Schlink K. Reduction of proteins during sample preparation and two-dimensional electrophoresis of woody plant samples. Proteomics 2006, 6: 1599–1605 Contribution: experiment design, two-dimensional electrophoresis, data analysis, writing the manuscript 2 Vâlcu CM, Schlink K. Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis. Proteomics 2006, 6: 4166–4175 Contribution: experiment design, two-dimensional electrophoresis, image and data analysis, writing the manuscript 3 Vâlcu CM, Vâlcu M. Reproducibility of two-dimensional electrophoresis at different replication levels. accepted for publication in Journal of Proteome Research Contribution: experiment design, two-dimensional electrophoresis, image and data analysis, writing the manuscript 4 Vâlcu CM, Lalanne C, Plomion C, Schlink K. Protein expression during the recovery from heat stress of Norway spruce (Picea abies) provenances from contrasting elevations. submitted Contribution: experiment design, two-dimensional electrophoresis, image and data analysis, preparing protein spots for mass spectrometry, analysis of mass spectrometry results, writing the manuscript 5 Vâlcu CM, Lalanne C, Müller-Starck G, Plomion C, Schlink K. Protein polymorphism between two Picea abies populations revealed by two-dimensional electrophoresis and tandem mass spectrometry. submitted Contribution: experiment design, two-dimensional electrophoresis, image and data analysis, preparing protein spots for mass spectrometry, analysis of mass spectrometry results, writing the manuscript 6 Vâlcu CM, et al. Local and systemic changes in European beech (Fagus sylvatica) proteome following infection with Phytophthora citricola. in preparation Contribution: experiment design, two-dimensional electrophoresis, image and data analysis, preparing protein spots for mass spectrometry, analysis of mass spectrometry results, writing the manuscript 4 Figures and Tables Index Figures 2.1-1 2-DE general protocol. 13 2.2.1-1 Response to and recovery from heat stress of Picea abies seedlings: experimental design. 16 2.2.1-2 Temperature profile and sampling time points. 16 2.2.2-1 Reaction of Fagus sylvatica seedlings to wounding: experimental design. 17 2.2.2-2 Reaction of Fagus sylvatica seedlings to infection with Phytophthora citricola: experimental design. 17 3.3-1 Infection experiment 1: symptoms of infection with Phytophthora citricola in Fagus sylvatica leaves. 21 3.3-2 Infection experiment 2: symptoms of infection with Phytophthora citricola in Fagus sylvatica roots. 21 4.1-1 Spot parameters with significant influence on spot volume variance. 23 4.2-1 Overview of plant reaction to heat induced oxidative stress. 25 4.3-1 Cluster analysis of Fagus sylvatica root proteins regulated following root infection with Phytophthora citricola and/or wounding. 26 4.3-2 Cluster analysis of Fagus sylvatica leaf proteins regulated following root infection with Phytophthora citricola and/or wounding. 27 4.3-3 Work model for the study of plant response to pathogen attack and wounding. 28 Tables 2.1-1 Optimisation of 2-DE protocols for Picea abies needles and roots and Fagus sylvatica 13 leaves and roots. 2.2.2-1 Overview of wound and infection experiments (Fagus sylvatica). 18 3.1-1 Optimised composition of extraction and focusing buffers. 19 5 List of abbreviations 2-DE two dimensional electrophoresis 2-ME 2-mercaptoethanol A anode AA acrylamide ASB 14 tetradecanoylamido propyl dimethylammonio propanesulphonate BPB bromophenolblue C cathode C7BzO 3-(4-heptyl) phenyl 3-hydroxypropyl dimethylammonio propanesulphonate C8Ø 4-octyl benzoyl amidopropyl dimethylammonio propanesulphonate CA carrier ampholyte DM dodecyl maltoside DTE dithioerythritol ET ethylene Glm general linear model HED hydroxyethyldisulphide HR hypersensitive response HS heat shock HSP heat shock proteins HSR heat shock response IAA iodoacetamide IEF isoelectric focusing IPG immobilised pH gradient ISR induced systemic resistence JA jasmonic acid NDSB non detergent sulphobetaines NDSB 256 N-phenyl-methyl-N,N-dimethyl ammonio propanesulphonate NP-40 Nonidet P-40 OG n-octyl β-D-glucopiranoside PAA polyacrylamide PTM post-translational modification SA salicylic acid SAR systemic acquired resistence sHSP small heat shock proteins SB 3-10 decyl dimethylammonio propanesulphonate TBP tributyl phosphine TCEP Tris (2-carboxyethyl) phosphine VOC volatile organic compounds 6 1 INTRODUCTION of the hydrological cycle as well as the increased frequency of extreme climatic events (extreme 1.1 Plant stress in the context of climate change temperatures and irregular spatial and temporal distribution of precipitations) are more severe in The stress concept in plants continental areas at middle and high latitudes and at high altitudes 10,11. The “stress” concept was founded by Hans Selye in 7 1936 , subsequently adapted for plant species and Effects of climate change upon natural formally defined as “any unfavourable condition or populations of a wide variety of species have substance that affects or blocks a plant’s already been registered. Latitudinal and altitudinal 8 metabolism, growth or development” . Stress shifts in the range of species distribution have factors induce a sequence of reactions grouped in been observed in several geographic regions12. A three phases: response, resistance and exhaustion, meta-analysis performed by Root et al.13 on 143 followed by a recovery phase after the removal of selected studies detected a statistically significant 8 the stressor . shift of species-characteristic spring events towards earlier dates with about 5 days per The distinctive feature that renders plants average decade within the last 50 years. Notable particularly vulnerable to changes in environmental exceptions were tree species for which the conditions is the fact that they are sessile. Since estimated average phenological shift was of only they cannot avoid non-optimal environmental 3 days13. conditions or escape environmental extremes through migration, as most animal species, plants Stress particularities in tree species have evolved a wide range of stress specific and unspecific mechanisms of resistance and tolerance. Life cycle related particularities of tree species Plants have also developed great acclimation and constitute disadvantages in the context of rapid adaptation capacities involving
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