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The Role of Mitochondrial Alternative Oxidase in Plant- Pathogen Interactions

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The Role of Mitochondrial Alternative Oxidase in Plant- Pathogen Interactions The Role of Mitochondrial Alternative Oxidase in Plant- Pathogen Interactions by Marina Cvetkovska A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Cell & Systems Biology University of Toronto © Copyright by Marina Cvetkovska 2012 1 The Role of Mitochondrial Alternative Oxidase in Plant-Pathogen Interactions Marina Cvetkovska Doctor of Philosophy Department of Cell & Systems Biology University of Toronto 2012 Abstract Alternative oxidase (AOX) is a non-energy conserving branch of the mitochondrial electron transport chain (ETC) which has been hypothesized to modulate the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria. The aim of the research presented herein is to provide direct evidence in support of this hypothesis and to explore the implications of this during plant-pathogen interactions in Nicotiana tabacum . We observed leaf levels of ROS and RNS in wild-type (Wt) tobacco and transgenic tobacco with altered AOX levels and we found that plants lacking AOX have increased levels of both NO and - mitochondrial O 2 compared Wt plants. Based on the results we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of electrons from the ETC to O 2 or nitrite. We characterized multiple responses of tobacco to different pathovars of the bacterial pathogen Pseudomonas syringae . These included a compatible response associated with necrosis (pv tabaci ), an incompatible response that included the hypersensitive response (HR) (pv maculicola ) and an incompatible response that induced defenses (pv phaseolicola ). We show that - the HR is accompanied by an early mitochondrial O 2 burst prior to cell death. Also, we found ii - that the appearance of HR and the appearance of the mitochondrial O 2 burst are delayed in transgenic plants lacking AOX. A similar delay is seen in transgenic plants treated with the complex III inhibitor antimycin A. In Wt plants, expression of Aox1a is suppressed during the HR response to pv maculicola despite the accumulation of signaling molecules known to induce Aox1a transcription. Also, MnSOD activity declined during the HR. We suggest that the mitochondrial ROS burst controlled by AOX and MnSOD is an important component for the induction and coordination of the HR during plant-pathogen interactions. iii Acknowledgments I am very thankful to my supervisor, Dr. Greg Vanlerberghe, for the guidance and support during my time in his lab. His enthusiasm and belief in my abilities have motivated me to continue the struggle even at times when the work seemed overwhelming. I think that his approach in providing me with just the right amount of guidance and independence has helped me to grow as a scientist. I would also like to thanks my supervisory committee members, Dr. Rene Harrison and Dr. Keiko Yoshioka, for their guidance in my research work and invaluable suggestions during the writing of my thesis. Finally, I would like to thank the examiners, Dr. Malcolm Campbell and Dr. Andrei Igamberdiev, for their involvement in my work. I would also like to express my gratitude to my colleagues in the lab. I would like to thank Dr. Sasan Amirsadeghi for introducing a very nervous first year grad student to the intricacies of lab work. Steven Wang and Melissa Cheung have been great friends to me both in and outside of the lab. I couldn't have done it without their help in both lab work and personal life. I would also like to thank all my other lab mates from the 2nd floor SRB, their friendship and humour have been the source of some of my fondest memories in grad school. I am also very grateful to my parents, Biljana and Cvetan, and to my sisters, Vedrana and Sara. Their encouragement has been more valuable to me than even they have suspected. Finally, I would like to thank my husband, David, who has learned more about plants in the past six years than he ever wanted. I could not have made it this far without his love and support. iv Table of Contents Abstract …………………………………………………………………………………………..ii Acknowledgements ……………………………………………………………………………...iv Table of Contents ……………………………………………………………………......……….v List of Tables …………………………………………………………………………………….ix List of Figures …………………………………………………………………………………….x List of Abbreviations ………………………………………………………………………......xiv List of Appendices ....................................................................................................................xviii CHAPTER 1: Mitochondrial signaling and homeostatic maintenance during plant- pathogen interactions: Literature review and thesis introduction ............................................1 Literature review..............................................................................................................................2 1.1 The plant mitochondrial electron transport chain..........................................................2 1.2 Non-phosphorylating pathways of the mETC...............................................................3 1.2.1 Alternative NAD(P)H dehydrogenases..........................................................4 1.2.2 Alternative oxidase.........................................................................................4 1.2.3 Uncoupling proteins........................................................................................8 1.3 The Pseudomonas syringae -plant model interaction...................................................10 1.3.1 Compatible plant-pathogen interactions.......................................................14 1.3.2.Incompatible plant-pathogen interactions.....................................................14 1.3.3 Non-host plant pathogen interactions...........................................................14 1.4 Programmed cell death in the plant immune response................................................15 1.4.1 Reactive oxygen species...............................................................................20 1.4.2 Reactive nitrogen species..............................................................................24 1.4.3 Hormonal signaling during biotic stress.......................................................28 1.5 The role of AOX during plant-pathogen interactions..................................................29 v Thesis introduction.........................................................................................................................31 CHAPTER 2: Materials and methods .......................................................................................35 2.1 Growth conditions........................................................................................................36 2.2 Imaging of cellular ROS and RNS...............................................................................37 2.3 SOD activity.................................................................................................................38 2.4 NO analysis..................................................................................................................39 2.5 H2O2 determination......................................................................................................39 2.6 Bacterial cultures and plant inoculations.....................................................................40 2.7 Leaf cell death and bacterial proliferation in planta ....................................................40 2.8 SA determination........................................................................................................41 2.9 RNA extraction and transcript determination.............................................................41 2.10 Mitochondria extraction and protein determination...................................................44 2.11 Chemical treatment of suspension cells and culture viability....................................46 2.12 DNA extraction..........................................................................................................46 2.13 Statistical analysis......................................................................................................47 CHAPTER 3: Alternative oxidase modulates leaf mitochondrial levels of superoxide and nitric oxide. ...................................................................................................................................48 3.1 Introduction..................................................................................................................49 3.2 Results..........................................................................................................................50 3.2.1 Various controls ensuring the validity of confocal images...........................50 3.2.2 Superoxide levels and SOD activity in transgenic plants with altered AOX levels............................................................................................................59 3.2.3 Nitric oxide levels in transgenic plants with altered AOX levels.................63 3.2.4 Hydrogen peroxide levels in transgenic plants with altered AOX levels....67 3.2.5 Peroxynitrite levels in transgenic plants with altered AOX levels..............67 vi 3.3 Discussion....................................................................................................................69 CHAPTER 4: Coordination of mitochondrial superoxide burst during the hypersensitive response to bacterial pathogen in Nicotiana tabacum ...............................................................74
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