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Gene Induction by Nitric Oxide (NO) in Arabidopsis Thaliana

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Gene Induction by Nitric Oxide (NO) in Arabidopsis Thaliana Lehrstuhl für Botanik der Technischen Universität München Gene Induction by Nitric Oxide (NO) in Arabidopsis thaliana Xi Huang Vollständiger Abdruck der von der Fakultät 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. E. F. Elstner Prüfer der Dissertation: 1. Univ.-Prof. Dr. E. Grill 2. Priv.-Doz. Dr. J. Durner Die Dissertation wurde am 25. 09. 2003 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 13. 11. 2003 angenommen. Finished in Institute of Biochemical Plant Pathology GSF - National Research Center for Environment and Health Under Supervision by PD Dr. Jörg Durner Index I Index I. Introduction........................................................................................................................... 1 1. Chemical basis for NO-mediated signaling effects................................................................ 2 1.1. Peroxynitrite formation ............................................................................................... 2 1.2. Formation of S-nitrosylation ....................................................................................... 3 1.3. Reaction with transition metals................................................................................... 3 2. NO signaling in mammalian systems..................................................................................... 4 2.1. Nitric oxide biosynthesis in mammals ........................................................................ 4 2.2. Regulation of gene expression by nitric oxide............................................................ 5 2.2.1. NO and DNA-methylation ................................................................................... 6 2.2.2. NO and transcription factors ................................................................................ 6 2.2.3. NO effects mRNA stability and translation ......................................................... 7 2.2.4. Regulation of posttranslational events by NO...................................................... 7 2.3. NO signaling and animal immune response................................................................ 8 3. NO signaling in plants.......................................................................................................... 10 3.1. Biosynthesis of nitric oxide in plants ........................................................................ 10 3.2. NO signaling in plant defense responses................................................................... 12 3.2.1. Mechanisms of plant defense responses............................................................. 12 3.2.2. The role of NO in plant defense responses ........................................................ 14 4. Goal of the work and the research strategy.......................................................................... 17 4.1. Simulation of NO burst in Arabidopsis plants and suspension cells......................... 17 4.2. Use of cDNA microarrays to study different gene expression.................................. 18 4.3. Verify and explain the gene induction ...................................................................... 18 II. Materials and Methods..................................................................................................... 20 1. Materials............................................................................................................................... 20 1.1. Plant materials........................................................................................................... 20 1.2. Chemicals.................................................................................................................. 20 1.3. Molecular biological Kits.......................................................................................... 22 1.4. Buffers and solutions................................................................................................. 22 Index II 1.5. Mediums.................................................................................................................... 25 1.6. Apparatus .................................................................................................................. 26 1.7. Consumed materials .................................................................................................. 27 2. Methods................................................................................................................................ 28 2.1. Growth condition of Arabidopsis plants and treatment with NO.............................. 28 2.2. Cell culture and NO treatment .................................................................................. 28 2.3. Microarray................................................................................................................. 28 2.3.1 PCR amplification and purification of target DNA ............................................ 29 2.3.1.1 Amplification of the target DNA ................................................................. 29 2.3.1.2. Purification of target DNA.......................................................................... 30 2.3.2 Array printing...................................................................................................... 31 2.3.3. Post-washing and blocking................................................................................. 32 2.3.4 Probe labeling...................................................................................................... 32 2.3.4.1 RNA extraction ............................................................................................ 32 2.3.4.2. cDNA synthesis........................................................................................... 34 2.3.4.3. Reaction Purification I: Removal of unincorporated aminoallyl-dUTP (aa- dUTP) and freeamines.............................................................................................. 34 2.3.4.4. Coupling aminoallyl-labeled cDNA to Cyanine dye ester......................... 35 2.3.4.5. Reaction purification II: removal of uncoupled dye ................................... 36 2.3.5. Pre-hybridization................................................................................................ 36 2.3.6. Hybridization...................................................................................................... 37 2.3.7. Post-washing and scanning ................................................................................ 37 2.3.8. Data collection and analysis............................................................................... 37 2.4. Northern blotting....................................................................................................... 38 2.4.1 Agarose/formaldehyde gelelectrophoresis .......................................................... 39 2.4.2 Transfer of RNA from gel to membrane............................................................. 39 2.4.3. Preparation of DIG-labeling DNA probe........................................................... 40 2.4.4. Hybridization...................................................................................................... 41 2.4.5. Washing, blocking and detection ....................................................................... 41 2.4.6. Designing specific primers for AOX genes and 12-oxo-phytodienoic acid reductase (OPR3) ......................................................................................................... 42 2.5. Cellular respiration.................................................................................................... 42 2.6. Cell death assay......................................................................................................... 43 2.7. Determination of SA, ethylene and JA...................................................................... 43 Index III 2.8. NO detected by fluorescent microscopes.................................................................. 43 2.9. NO detection by electron paramagnetic resonance (EPR)........................................ 44 III. Results............................................................................................................................... 45 1. Preparation of the cDNA used for spotting.......................................................................... 45 2. Microarray analysis of transcripts of suspension cells after NO treatment.......................... 47 2.1 Data analysis and normalization ................................................................................ 48 2.2. Dynamics of gaseous NO-inducible gene expression............................................... 49 3. Identification of the alternative oxidase (AOX) as NO counteracting principle.................. 52 3.1. Northern blot verify the AOX induction by NO ....................................................... 52 3.2. Identification of other AOX homolog in Arabidopsis .............................................. 54 3.3. Identification of the transcript of AOX1a in mutants ............................................... 56 3.4. Determination of the respiration after NO treatment ................................................ 57 3.5. Identify
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