Cluster analysis and comparison of various chloroplast and nuclear transcriptomes in Arabidopsis thaliana [Elektronische Ressource] / vorgelegt von Won Kyong Cho

Cho Won Kyong

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 3
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

             

   

Cluster Analysis and Comparison of Various Chloroplast and Nuclear Transcriptomes inArabidopsis thaliana  

Dissertation  zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München

 

    vorgelegt von Won Kyong Cho aus Seoul, Korea   2007

 

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                            Erstgutachter : PD Dr. J. Meurer

Zweitgutachter : Prof. Dr. R.G. Herrmann

Datum der mündlichen Prüfung: 22. 06. 2007

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Contents  ABBREVIATIONS ....................................................................... - 5 -  1 INTRODUCTION ...................................................................... - 7 -  1.1 Origin of the Plastid Genome................................................................................... - 7 - 1.2 Regulation of Plastid Gene Expression at the Transcriptional Level.................. - 8 - 1.3 Regulation of Plastid Gene Expression at the Post-Transcriptional Level.......... - 9 - 1.4 Microarray Techniques.......................................................................................... - 10 - 1.5 Gene Expression Analysis in Higher Plants Using Microarray Technique....... - 12 - 1.6 Aim of the Project................................................................................................... - 12 - 2 MATERIALS AND METHODS ............................................. - 15 -  2.1 Materials.................................................................................................................. - 15 - 2.1.1 Chemicals, Enzymes, Radioactive Substances, and Devices.. - 15 -...................... 2.1.2 Source ofArabidopsisMutants, Phenotypes, and Growth Conditions........ - 15 - 2.1.3 Media, Solutions and Buffers.......................................................................... - 18 - 2.1.4 Softwares........................................................................................................... - 19 - 2.1.5. Oligonucleotides used for the generation and spotting macroarray gene probes......................................................................................................................... - 20 - 2.2 Methods.................................................................................................................... - 24 - 2.2.1 RNA Isolation and Gel Blot Analysis............................................................. - 24 - 2.2.2 Preparation of Macroarray Filters................................................................. - 24 - 2.2.3 Hybridization of Labelled cDNAs to Macroarray Filters............................ - 25 - 2.2.4 Normalization and Statistical Analysis.......................................................... - 25 - 2.2.5 Microarray Data Analysis - 26 -............................................................ ... ................ 2.2.6 Promoter Analysis............................................................................................ - 26 - 3 RESULTS.................................................................................. - 27 -  3.1 Plant Growth and Mutant Phenotypes.... - 27 -.............................................................. 3.2 Establishment of Plastid Macroarrays 27 -................... -............................................... 3.3 Expression Profiling of Plastid Genes under Various Biological Conditions.... 29 -- 3.4 Cluster Analyses of Plastid Genes deduced from 89 Transcriptomes................ - 30 - 3.5 Identification of Mutants Affected in the Chloroplast mRNA Metabolism...... - 37 - 3.6 Evaluation of Nuclear and Plastid Gene Expression using Affymetrix Microarray Data................................................................................................................................. - 39 -

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3.7 Use of Microarrays for the Evaluation of Plastid Gene Expression in Various ArabidopsisMutants........................................................................................... - 40 -........... 3.8 Use of Microarrays for the Evaluation of Plastid Gene Expression under Various Stress Conditions........................................................................................................... - 42 - 3.9 Use of Microarrays for the Evaluation and Comparison of Nuclear and Plastid Gene Expression under Various Stress Conditions................................................... - 45 - 3.10 Distribution of Plastid and Nuclear Gene Expression Ratios under Different Biological Conditions.................................................................................................... - 49 - 3.11 Plastid Gene Expression in Response to Different Light Qualities during Early Seedling De-etiolation................................................................................................... - 51 - 4 DISCUSSION............................................................................ - 57 -  4.1 Analysis of Chloroplast Transcriptomes............................................................... - 57 -  4.2 Hierarchical Clustering of Plastid Mutant Transcriptomes Identified Two Distinguishable Signatures and Novel Mutants Impaired in mRNA Metabolism.. - 57 - 4.3 Hierarchical Clustering of Plastid Genes in Mutants of Chloroplast Functions Identified Two Transcriptionally Determined Gene Clusters.................................. - 58 - 4.4 Nuclear Genes for Plastid Components Displayed Dynamical Gene Expression Patterns, Eight Major Co-Regulated Clusters and 13 Transcriptome Groups....... - 60 - 4.5 Phytochrome Signalling Suppresses Plastid Gene Expression during Early Seedling De-Etiolation before Nuclear Genes Start to Respond............................... - 61 - SUMMARY.................................................................................. - 64 -  REFERENCES ............................................................................ 65 -  - ACKNOWLEDGMENTS........................................................... - 77 -  CURRICULUM VITAE ............................ - 78 -  ... .............................. PUBLICATIONS............................................... ........ - 79 -................ ..        

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 ABBREVIATIONS   microeinstein (1 E = 1 mol of photons) adenosine 5′ohpsahet-rtpi base pairs blue light complementary DNA curie counts per minute deoxyribonucleic acid 2-deoxycytidine 5-triphosphate deoxynucleoside triphosphates ethylenediaminetetraacetic acid ethyl methanesulfonate expressed sequence tags gravity force, gramme far-red light high chlorophyll fluorescence kilobases locally weighted regression megabases 2-Morpholinoethanesulfonic acid 3-[N-Morpholino]propanesulfonic acid messenger RNA murashige and skoog medium national center for biotechnology information nuclear encoded RNA polymerase open reading frame pulse amplitudemodulated fluorometer polymerase chain reaction

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µE ATP bp B light cDNA Ci cpm DNA dCTP dNTPs EDTA EMS ESTs g FR light hcf kb Lowess Mb MES MOPS mRNA MS NCBI NEP ORF PAM PCR

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PEP Phy PSI PSII qP R light RNA rpm rRNA RT-PCR S SD SDS SOM T-DNA Tm Tris tRNA U UTR UV v/v w/v 

plastid encoded RNA polymerase phytochrome photosystem I photosystem II photochemical chlorophyllafluorescence quenching red light ribonucleic acid revolutions per minute ribosomal RNA reverse transcription PCR svedberg unit standard deviation sodium dodecyl sulfate self organizing map transferred DNA annealing temperature tris-(hydroxymethyl)-aminomethane transfer RNA unit, enzyme activity untranslated region ultra violet volume per volume weight per volume

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1 INTRODUCTION  1.1 Origin of the Plastid Genome  The chloroplast evolved as a result of an endosymbiotic event in which a cyanobacterial ancestor was taken over by a eukaryotic cell. Although most chloroplast genes have been lost or transferred to the nucleus and the majority of plastid proteins is encoded by nuclear genes, the organelle still retained the coding capacity for a number of genes and possesses its own gene expression machinery (Race et al., 1999) (Figures 1 and 2). Due to its endocytobiotic parentage, chloroplast gene expression represents a unique chimeric system assembled from multiple origins (Barkan and Goldschmidt-Clermont, 2000; Monde et al., 2000; Rochaix, 2001). 

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Figure 1.Endosymbiotic Rearrangement of Genes and Proteins in Plants. The estimated numbers and the localisation of genes and proteins of endosymbiotic origin. Dotted lines, genes of endosymbiotic origin. Solid lines: localisation of nuclear encoded proteins. Green: genes and proteins of cyanobacterial origin; black: genes and proteins of other origin.

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 Figure 2. Functional Phylogenetic Model of the Photosynthetic Membrane.  The four major complexes are shown. A fifth minor abundant complex, the NADH- dehydrogenase complex, is also present in the thylakoid membrane (not shown). Yellow and green colours indicate nuclear and plastid encoded proteins for photosynthetic complexes (Race et al., 1999).   Plastid genes are embedded into regulatory networks that enable an adaptive and developmentally dependent chloroplast biogenesis at various levels (Figure 2; Table 1). An intriguing number of plastid transcriptional and posttranscriptional events were acquired in the result of endosymbiosis, which are not found at that extent in currently living cyanobacteria. Knowledge about this regulation is not only increasingly relevant for studying phylogenetic and ontogenetic aspects of chloroplast biogenesis but also for applying transplastomic approaches, since plastomes of several model plants as well as of agriculturally important plants will be genetically engineered in the near future (Bock, 2006). Numerous nuclear-encoded factors with yet unidentified functions play a crucial role in the regulation of the complex chloroplast transcript homeostasis.  1.2 Regulation of Plastid Gene Expression at the Transcriptional Level  Transcription rates depend on light, tissue- and cell-type. They also undergo endogenous circadian rhythms and possibly redox regulation (Allison, 2000). The activity of the plastid- encoded RNA polymerase (PEP) is regulated by nuclear-encoded sigma factors which are involved in the global and specific environmental and developmental dependent as well as tissue-specifc regulation of plastid transcription (Tanaka et al., 1996; Allison, 2000; Privat et al., 2003; Ichikawa et al., 2004; Favory et al., 2005; Zghidi et al., 2007). Sigma factors

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