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Table of contents
1 INTRODUCTION
2 CURRENT STATE OF KNOWLEDGE
2.1 Role of salicylic acid in plants
2.1.1 Systemic acquired resistance and SA
2.1.1.1 SA signalling pathway
2.1.1.2 Studies of SAR transcriptome
2.1.2 Mutants with altered SA/SAR signalling
2.1.2.1 Mutants pointing to lipid-based signalling in SAR
2.1.2.2 Mutants affected in SA synthesis
2.1.2.3 SA signalling in redox mutants
2.1.2.4 NPR1-dependent and –independent pathways
2.1.2.5 Connections and cross-talks
2.1.2.6 Metabolic costs of induced resistance
2.1.3 SA in other physiological contexts
2.1.3.1 Temperature stress
2.1.3.2 Oxidative stress
2.1.3.3 Plant development
2.2 Phospholipid signalling in plants
2.2.1 Key phospholipid signalling molecules
2.2.1.1 Phosphoinositides
2.2.1.2 PA and DGPP
2.2.1.3 Free fatty acids and lyso-phospholipids
2.2.2 Key enzymes in phospholipid signalling
2.2.2.1 PI 3-kinase
2.2.2.2 PI 4-kinase
2.2.2.3 PIP 5-kinases
2.2.2.4 Phospholipase C
2.2.2.5 DAG- and PA-kinase
2.2.2.6 Phospholipase D
2.2.2.7 Phospholipase A
2.2.3 Possible cellular targets of phospholipid signals
2.2.3.1 FYVE domain
2.2.3.2 PH domain
2.2.3.3 C2 domain
2.2.3.4 Other phospholipid-binding domains
3 MATERIALS AND METHODS
3.1 Materials
3.1.1 Chemicals and kits
3.1.2 Plant materials
3.1.3 Characterization of T-DNA insertion mutants
3.2 Characteristics of cell suspensions
3.2.1 Growth curve
3.2.2 Viability staining
3.3 Phospholipid analysis
3.3.1 SA treatment and phospholipid analysis
3.3.2 Analysis of PI species by RP-HPLC
3.4 Gene expression analyses
3.4.1 RNA extraction for semiquantitative RT-PCR and microarray experiments
3.4.2 RNA extraction for QRT-PCR
3.4.3 Semiquantitative RT-PCR analysis
3.4.4 QRT-PCR analysis
3.4.5 Transcriptome studies
3.4.6 Statistical analysis of microarray data
3.5 Data deposition
4 RESULTS
4.1 Introductory experiments leading to characterization of the model system
4.1.1 Non-lethal and effective SA concentration
4.1.2 Marker genes of the SA pathway
4.1.3 Radiolabelling of phospholipids in vivo
4.2 SA activates PI 4-kinase in vivo
4.2.1 Radiolabelling of phospholipids during SA treatment
4.2.2 Characteristics of the SA-induced changes in labelled phospholipids
4.2.3 SA-induced PIP is PI(4)P
4.2.4 PI decrease can be impaired by inhibitors of type III PI 4-kinase
4.2.5 Phosphorylation events are involved in the activation of PI 4-kinase
4.2.6 Ca2+ influx in response to SA
4.2.7 Expression of genes involved in the phosphoinositide metabolism during SA treatment
4.3 SA transcriptome is partially regulated by a W30-sensitive pathway
4.3.1 SA induces changes in the transcriptome of Arabidopsis
4.3.2 Effect of wortmannin on the SA-regulated transcriptome
4.3.3 Common cis-elements in the promoters of SA-regulated genes
4.3.4 Expression of selected marker genes of the W30-sensitive pathway in the T-DNA mutants of PI 4-kinase β
4.4 SA activates PLD in vivo
4.4.1 Expression of several SA-regulated genes is regulated by PLD
4.4.2 PLD intervenes in the early stages of SA treatment
4.4.3 Exogenous PA application did not reverse the n-butanol inhibition of SA response
4.5 SA transcriptome is partially regulated by the PLD pathway
4.5.1 Identification of PLD-regulated genes in the SA transcriptome
4.5.2 Common cis-elements in the promoters of PLD-regulated genes in response to SA
4.5.3 Expression of PR1 and WRKY38 in the T-DNA mutants of several PLDs
4.6 Overlap of W30-sensitive and PLD-regulated SA transcriptomes
5 DISCUSSION
5.1 Arabidopsis cell suspensions are an admissible model to study SA signalling
5.2 Activation of a PI 4-kinase as an early response to SA
5.3 Phosphorylation events precede the PI 4-kinase activation in response to SA
5.4 Ca2+ signalling is not involved in the early SA response
5.5 Protein synthesis differentially affects the SA response
5.6 SA-regulated transcriptome
5.7 Effects of wortmannin on the SA-responsive transcriptome
5.8 PI4Kβ1 may be involved in the SA response
5.9 PLD influences SA-regulated gene expression
5.10 Effects of PLD on the SA-responsive transcriptome
5.11 PI(4,5)P2-dependent PLDs modulate the SA response
5.12 PI 4-kinase and PLD act synergistically in the SA signalling pathway
6 CONCLUSIONS
7 ABBREVIATIONS
8 LITERATURE CITED
9 SUPPLEMENTAL DATA
