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Functional Characterization of Mitochondrial Stress Response in Arabidopsis Thaliana

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Functional Characterization of Mitochondrial Stress Response in Arabidopsis Thaliana Functional characterization of mitochondrial stress response in Arabidopsis thaliana Botao Zhang School of Chemistry and Biochemistry and the ARC Centre of Excellence in Plant Energy Biology This thesis is presented for the degree of Doctor Philosophy of the University of Western Australia April 2012 Coordinating supervisor: Prof. James Whelan Co-supervisor: Dr. Olivier Van Aken Declaration The research presented in this thesis is my own work unless otherwise stated. This work was carried out in the Australian Research Council Centre of Excellence in Plant Energy Biology at the University of Western Australia. The material presented in this thesis has not been submitted for any other degree. Botao Zhang April 2012 I Acknowledgement First and foremost I would like to thank my supervisor Jim Whelan for all his expertise, guidance and untiring support over the past few years. I really appreciate the enthusiastic help you give me during and after my PhD study. I would also like to thank all of the members from the Whelan lab for making a great working environment. My sincere gratitude must go to Dr Olivier Van Aken. Thanks for your endless help, encouragement and advice in both science and life. It will be the invaluable part kept in my memory. I would also like to thank other members of the Whelan lab. Special mentions must go to Chris, Aneta and Estelle for the professional advice and the Chinese community Yan, Lin, Wendy and Sophia for the hometown friendship. I want to thank everyone else from the Centre of Plant Energy Biology, as they are so nice and it really has been a great place to work. I would like to thank my family who have supported me throughout my life. I can’t ask for more from you. Lastly, particular appreciation goes to my wife Wona for the unconditional love and support. Thank you for tiding over the difficult years together with me. II Publications arising from this thesis Van Aken, O., Zhang, B., Carrie, C., Uggalla, V., Paynter, E., Giraud, E., and Whelan, J. (2009). Defining the mitochondrial stress response in Arabidopsis thaliana. Mol Plant 2, 1310-1324. (Chapter 2) Zhang, B., Carrie, C., Ivanova, A., Narsai, R., Murcha, M.W., Albrecht, V., Pogson, B., Giraud, E., Van Aken, O., and Whelan, J. (2012). LETM2 is required for translation of mitochondrial encoded transcripts and is controlled by maternal epigenetic pathways in Arabidopsis thaliana. Submitted to the Plant Cell. (Chapter 3) Zhang, B., De Clerq, I., Van Breusegem, F., Whelan, J., and Van Aken, O. (2012). The AAA ATPase AtBCS1 is an outer mitochondrial membrane protein that affects pathogen resistance in Arabidopsis thaliana. In preparation. (Chapter 4) III Other publications Kühn, K., Carrie, C., Giraud, E., Wang, Y., Meyer, E.H., Narsai, R., des Francs- Small, C.C., Zhang, B., Murcha, M.W., and Whelan, J. (2011). The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. Plant J 67, 1067- 1080. Duncan, O., Taylor, N.L., Carrie, C., Eubel, H., Kubiszewski-Jakubiak, S., Zhang, B., Narsai, R., Millar, A.H., and Whelan, J. (2011). Multiple lines of evidence localise signalling, morphology and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis thaliana. Plant Physiology 157(3): 1093-1113. Van Aken, O., Zhang, B., and Whelan, J. (2012). WRKY transcription factors co-regulate genes encoding mitochondrial and chloroplast proteins in Arabidopsis thaliana.In submittion to the Plant Physiology. IV Abstract Plants, because of their immobile nature, are continually exposed to a variety of extreme environmental conditions i.e. stresses. Stresses have adverse effects on plant growth, development and seed production. Nearly all biotic and abiotic stresses disrupt the metabolic balance of cells, resulting in osmotic stress and enhanced production of reactive oxygen species (ROS) (Mittler et al., 2004). Mitochondria are one of the major sources of ROS and a major target of oxidative stress, and are vital for maintaining the viability during biotic and abiotic stress. Previous studies showed that there are a number of mitochondrial proteins that are induced by different treatments directly targeted to mitochondria (Clifton et al., 2006). However, the response of mitochondria to stress and the roles mitochondria play under stress are still not very clear. In this study, the functional characterization of mitochondrial stress response was conducted. By combining a list of 1196 Arabidopsis thaliana genes that putatively encode mitochondrial proteins with 16 publicly available microarray datasets under stress conditions, 45 nuclear encoded proteins were defined as mitochondrial stress-responsive proteins. Furthermore, the mitochondrial localization of these proteins was tested using green fluorescent protein (GFP) fusion assays. In total 26 proteins could be confirmed to be targeted to mitochondria and were thus considered to be mitochondrial stress responsive proteins. Two of the mitochondrial stress responsive genes were selected for functional characterization. One of these proteins, AtLETM1 (leucine zipper-EF-hand-containing transmembrane protein), was investigated using T-DNA insertion lines. Analysis revealed the presence of a paralog in Arabidopsis. Inactivation of either AtLETM1 or AtLETM2 did not result in any severe deleterious growth phenotypes, while inactivation of both genes was lethal. However, plants that are hemizygous for AtLETM2 and homozygous for AtLETM1 (letm1 (-/-) LETM2 (+/-)) showed a retarded growth phenotype during early seedling growth. Studies revealed that a functional maternal AtLETM2 allele in the absence of V AtLETM1 was required for viability and was shown that the expression of paternal AtLETM2 allele was silenced during early embryogenesis. The translation rates in mitochondria of letm1 (-/-) LETM2 (+/-) plants were decreased, resulting in plants with reduced amounts of the D-subunit of mitochondrial ATP synthase and cytochrome c. The letm1 (-/-) LETM2(+/-) plants also displayed lower levels of ATP, increased total ascorbate levels and increased resistance to drought stress. These results show that AtLETM activity is essential for mitochondrial translation and its expression is under maternal control during early seed development. The second protein that was investigated was AtBCS1, an AAA-type ATPase. Subcellular localization assays confirmed its location in mitochondrial outer membrane. Phylogenetic and structural analysis indicated functional divergence between the plant BCS1 proteins and animal and fungal homologs, which are located on the mitochondrial inner membrane. The expression of AtBCS1 was highly increased in a set of MAPK signaling cascade mutants and by treatments of various biotic elicitors. The knockdown transgenic plants with decreased expression of AtBCS1 did not show obvious growth differences compared to wild type under normal and stress conditions. However, the overexpression transgenic plants of AtBCS1 displayed compressed rosette with curled leaves, and increased sensitivity to pathogen attack, suggesting that AtBCS1 is involved in biotic stress responses. VI Abbreviation × g times the force of gravity AMPS ammonium persulphate AOX alternative oxidase $73Į mitochondrial ATP synthase alpha subunit $73ȕ mitochondrial ATP synthase beta subunit BCS protein involved in cytochrome bc1 assembly BN-PAGE blue native polyacrylamide gel electrophoresis BP border primer BSA bovin serum albumin COX Cytochrome Oxidase Cyt c Cytochrome C DAB diaminobenzidine ddH2O double deionised water DMSO dimethyl sulfoxide ETC Electron transport chain Fv/Fm maximal quantum yield Gent gentamicin GFP Green fluoresence protein HSP heat shock proteins IPTG iVRSURS\Oȕ-D-1-thiogalactopyranoside Kan kanamycin VII LP left primer MES 2-(N-morpholino)ethanesulfonic acid MS Murashige & Skoog NBT nitroblue tetrazolium PCR polymerase chain reaction PK proteinase K PMSF phenylmethylsulfonyl fluoride psi pounds per square inch PVDF polyvinylidene fluoride PVP polyvinylpyrrolidone Redox reduction/oxidation Rif rifampicin ROS reactive oxygen species RP right primer RISP Rieske iron sulphur cluster protein RT-PCR reverse transcription PCR SA salicylic acid SDH succinate dehydrogenase SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SDW sterile double deionised water TCA tricarboxylic acid T-DNA transfer-DNA VIII Tris tris(hydroxymethyl)aminomethane TEMED N,N,N',N'-Tetramethylethylenediamine UCP uncoupling protein UQ ubiquinones UQH2 ubiquinol UTR untranslated region x-Gluc 5-bromo-4-chloro-3-indolyl-b-glucuronic acid IX Table of Contents Declaration...........................................................................................................I Acknowledgement...............................................................................................II Publications arising from this thesis...................................................................III Other publications............................................................................................. IV Abstract.............................................................................................................. V Abbreviation..................................................................................................... VII Table of Contents.................................................................................................i Chapter 1 Introduction ........................................................................................1
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