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Table of contents
CHAPTER 1 GENERAL INTRODUCTION
General overview of energy conversion in plants
1.1 ROS in plants
1.1.1 ROS: definition
1.1.2 ROS generation
1.1.2.1 ROS generation and compartmentation
1.1.2.2 Stress and ROS
1.1.3 ROS signaling
1.1.3.1 ROS and photosynthesis
1.1.3.2 ROS and redox homeostasis
1.1.3.3 ROS and Mitogen activated protein kinase (MAPK) 12
1.1.3.4 ROS and photohormones
1.1.3.5 Other components involved in ROS signaling
1.2 ROS processing
1.2.1 Catalase
1.2.2 Ascorbate and glutathione in ROS metabolism
1.2.2.1 Glutathione in plants
1.2.2.2 Ascorbate in plants
1.2.2.3 Ascorbate glutathione pathway
1.2.2.4 Other pathways of ROS processing
1.2.2.5 NADPH linked reaction in plants
1.2.2.5.1 Glucose 6 phosphate dehydrogenase
1.2.2.5.2 Isocitrate dehydrogenase
1.2.2.5.3 Nonphosphorylating glyceraldehyde 3 phosphate dehydrogenase
1.2.2.5.4 NADP malic enzyme
1.3 Lesion mimic mutants in plants
1.4 Arabidopsis: a model to aid quick progress in understanding plant function
1.5 Aims of the project
CHAPTER 2 THE FUNCTION OF SPECIFIC CATALASES IN PLANTS
2.1 Introduction
2.2 Material and methods
2.2.1 Plant material
2.2.2 Growth conditions and sampling
2.2.3 Enzyme activities
2.2.4 Measurements of transcript abundance
2.2.5 Ascorbate, glutathione and ROS assays
2.3 Results
2.3.1 CAT expression and activity in roots
2.3.2 Root and seed phenotypes in CAT mutants
2.3.3 CAT2 and CAT3 function in leaves
2.4 Discussion
2.4.1 Decreased root growth is specific to cat2 and is a secondary effect
2.4.2 The enigmatic roles of non–photorespiratory CATs in Arabidopsis
2.4.3 Growth day length affects oxidative signaling independent of oxidative stress duration
CHAPTER 3 EFFECT OF A SPECIFIC ISOFORM OF GLUCOSE–6–PHOSPHATE DEHYDROGENASE ON H2O2–INDUCED SA SIGNALING: A GENETIC SCREEN FOR REVERTANT LINES
3.1 Introduction
3.2 Results
3.2.1 The evaluation of mutagenesis efficiency
3.2.2 The revertant screen
3.2.3 Backcross to cat2 g6pd5 and segregation analysis
3.2.4 Identification of causal mutations
3.3 Discussion
CHAPTER 4 ANALYSIS OF THE ROLES OF MONODEHYDROASCORBATE REDUCTASES IN H2O2 METABOLISM USING GENE–SPECIFIC MUTANTS
4.1 Introduction
4.2 Results
4.2.1 Identification of mdhar single mutants
4.2.2 MDHAR transcripts in response to intracellular oxidative stress
4.2.3 Impact of mdhar mutations on cat2–triggered lesion formation and phytohormone signaling
4.2.4 Impact of the loss of MDHAR functions on leaf redox status
4.3 Discussion
CHAPTER 5 GENERAL CONCLUSION AND PERSPECTIVES
5.1 Conclusions
5.1.1 The function of specific CAT isoforms
5.1.2 Effect of G6PD5 on the H2O2–induced SA signaling pathway
5.1.3 Functions of specific MDHAR isoforms in response to oxidative stress
5.2 Perspectives
5.2.1 Specificity of CAT functions and interactions between oxidative signaling and day length
5.2.2 Further analysis of revertant mutations that allow lesion formation in cat2 g6pd5
5.2.3 MDHAR isoforms in responses to H2O2
5.2.4 A functional link between G6PD5 and MDHAR2 in H2O2 signaling?
CHAPTER 6 MATERIALS AND METHODS
6.1 Plant materials and growth conditions
6.1.1 Plant materials
6.1.2 Growth and sampling
6.2 Methods
6.2.1 Phenotypic analysis and lesion quantification
6.2.2 DNA extraction and plant genotyping
6.2.3 RNA extraction and transcripts analysis
6.2.4 Antioxidative enzyme activity measurements
6.2.4.1 Extraction
6.2.4.2 Activity assay
6.2.5 Metabolite analysis
6.2.5.1 Glutathione and ascorbate assay by plate reader
6.2.5.1.1 Extraction
6.2.5.1.2 Glutathione analysis
6.2.5.1.3 Ascorbate analysis
6.2.5.2 Total SA assay by High Performance Liquid Chromatography (HPLC)
6.2.6 ROS visualization in roots
6.2.7 EMS screen
6.2.7.1 EMS mutagenesis
6.2.7.2 Phenotype screen
6.2.7.3 Backcross with cat2 g6pd5
6.2.7.4 Sample collection for sequencing
6.2.8 Nuclear DNA isolation for sequencing
6.2.9 Statistical analysis
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