Max Planck-Institut für Molekulare Pflanzenbiologie und Universität Potsdam Arbeitsgruppe Prof. Dr. Bernd Mueller-Roeber Unraveling the ORE1 Regulon in Arabidopsis thaliana: Molecular and Functional Characterization of Up- and Down-stream Components Dissertation zur Erlangung des akademischen Grades “doctor rerum naturalium” (Dr. rer. nat.) in der Wissenschaftsdisziplin “Molekularbiologie” eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam von Lilian Paola Matallana-Ramírez Potsdam, den 11.04.2012 Published online at the Institutional Repository of the University of Potsdam: URL http://opus.kobv.de/ubp/volltexte/2012/6264/ URN urn:nbn:de:kobv:517-opus-62646 http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-62646 Lilian Paola Matallana-Ramírez Dedicated to my beloved deceased grandma Graciela “-Try not to become a man of success but rather to become a man of value”. A. Einstein ii Contents Summary Leaf senescence is an active process required for plant survival, and it is flexibly controlled, allowing plant adaptation to environmental conditions. Although senescence is largely an age-dependent process, it can be triggered by environmental signals and stresses. Leaf senescence coordinates the breakdown and turnover of many cellular components, allowing a massive remobilization and recycling of nutrients from senescing tissues to other organs (e.g., young leaves, roots, and seeds), thus enhancing the fitness of the plant. Such metabolic coordination requires a tight regulation of gene expression. One important mechanism for the regulation of gene expression is at the transcriptional level via transcription factors (TFs). The NAC TF family (NAM, ATAF, CUC) includes various members that show elevated expression during senescence, including ORE1 (ANAC092/AtNAC2) among others. ORE1 was first reported in a screen for mutants with delayed senescence (oresara1, 2, 3, and 11). It was named after the Korean word “oresara,” meaning “long-living,” and abbreviated to ORE1, 2, 3, and 11, respectively. Although the pivotal role of ORE1 in controlling leaf senescence has recently been demonstrated, the underlying molecular mechanisms and the pathways it regulates are still poorly understood. To unravel the signaling cascade through which ORE1 exerts its function, we analyzed particular features of regulatory pathways up-stream and down-stream of ORE1. We identified characteristic spatial and temporal expression patterns of ORE1 that are conserved in Arabidopsis thaliana and Nicotiana tabacum and that link ORE1 expression to senescence as well as to salt stress. We proved that ORE1 positively regulates natural and dark-induced senescence. Molecular characterization of the ORE1 promoter in silico and experimentally suggested a role of the 5’UTR in mediating ORE1 expression. ORE1 is a putative substrate of a calcium-dependent protein kinase named CKOR (unpublished data). Promising data revealed a positive regulation of putative ORE1 targets by CKOR, suggesting the phosphorylation of ORE1 as a requirement for its regulation. Additionally, as part of the ORE1 up-stream regulatory pathway, we identified the NAC TF ATAF1 which was able to transactivate the ORE1 promoter in vivo. Expression studies using chemically inducible ORE1 overexpression lines and transactivation assays employing leaf mesophyll cell protoplasts provided information on target genes whose expression was rapidly induced upon ORE1 induction. First, a set of target genes was established and referred to as early iii Lilian Paola Matallana-Ramírez responding in the ORE1 regulatory network. The consensus binding site (BS) of ORE1 was characterized. Analysis of some putative targets revealed the presence of ORE1 BSs in their promoters and the in vitro and in vivo binding of ORE1 to their promoters. Among these putative target genes, BIFUNCTIONAL NUCLEASE I (BFN1) and VND-Interacting2 (VNI2) were further characterized. The expression of BFN1 was found to be dependent on the presence of ORE1. Our results provide convincing data which support a role for BFN1 as a direct target of ORE1. Characterization of VNI2 in age-dependent and stress-induced senescence revealed ORE1 as a key up-stream regulator since it can bind and activate VNI2 expression in vivo and in vitro. Furthermore, VNI2 was able to promote or delay senescence depending on the presence of an activation domain located in its C-terminal region. The plasticity of this gene might include alternative splicing (AS) to regulate its function in different organs and at different developmental stages, particularly during senescence. A model is proposed on the molecular mechanism governing the dual role of VNI2 during senescence. iv Contents Acknowledgments During these past few years I have one of the most enriching times of my entire life. I growth as a science person, I proved my capacities, my patient, my limits and my self-confidence. I learned the real value of my friends, my family and the simple things of life that I left in my country Colombia. The most valuable knowledge is not only the one that I learn as a molecular biologist is the one that help me to improve my life to become a better person and reinforces my moral and my ethics. Nevertheless, none of this valuable life lessons would have been possible without the friendship and encourage of all my friends, family and supervisors. I would like to acknowledge my Ph.D supervisors, Prof Dr. Bernd Mueller-Roeber and Dr. Salma Balazadeh for giving me the opportunity to make my Ph.D thesis in the group and introduce me in the fascinating world of plant molecular biology, let me grow as a scientist. I want to thank the DAAD (Akademischer Austausch Dienst) for the financial support, the opportunity to learn another language and the facilities to integrate me into the German culture. I am heartily thankful to the DAAD representative for Latin America, Arpe Caspary for his friendship, for believing in me and my capacities. I wish also to thank the former Colombia referat Mrs. Silke Hamacher for her efficient work and nice attitude. I want to thank exclusively Dr. Judith Lucia Gómez-Porras. She careful read and editorial assisted my thesis. She established with me nutritive scientific discussions and her criticisms, support and humor were the most powerful inputs to complete successfully the final level of my Ph.D. I want to thanks Dr. Alex Ivakov and Matt Neville to point key aspects to improve the document. I want to thanks Dr. Francesco Licausi for stimulating discussions of the experiments, improvements on the document and his unconditional friendship. I received thoughtful suggestions, discussions and warm spiritual support from my Ph.D committee members Prof. Dr. Ingo Dreyer and Dr. Dirk Hincha. All of them directed me become a good scientist. I would specifically like to thank Dr. Eugenia Maximova who always takes the time to help me in microscopy, Dr. Karin Köhl and her Greenteam especially Linda Bartetzko, Katrin Seehaus, Sven Roigk, Helga Kulka, Britta Hausmann and Katrin Lepa for taking excellent care of plants and transformations and giving me always a smile and good advices; Josef v Lilian Paola Matallana-Ramírez Bergstein for the excellent photography work and nice conversations. Many thanks go also to the IT team; the administration team and the media kitchen for making my time at the MPI-Golm better and efficient. I want to thank all members of the Molecular Biology Department at the University of Potsdam especially Mathias Christoph, Eike Kamman, Dr. Dagmar Kupper, Antje Scheneider, Karina Schulz, Radoslaw Lukoszek, Dr. Minh Hung Nguyen and Ines Nowak for guiding me and teach me key aspects for my daily lab work. Also thanks for their friendschip and be always open to help me. I also want to thank Nooshin Omranian, Mamoona Rauf, Arif Muhammad, Prashant Garapati and Amin Omidbakhshfard for their support. My especial appreciation goes to my collaborators; Prof. Dr. Amnon Lers (Volcani Center, Israel), Dr.Gang-Ping Xue (CSIRO Plant Industry, Australia) and Prof. Dr. Wolfgang Dröge- Laser (Julius-Maximilians-Universität Würzburg) for excellent discussions and improvement on the article. I also keep in my hearth grateful my friends Daan Weits, Dr. Federico Giorgio, Anna Flis, Monika Chodasiewicz, Asdrubal Burgos, Rodrigo Caroca, Ignacia Bustos, Susana Salem, Dr. Sonia Osorio, Dr. Clara Sanchez, Phuong Anh Pham, Dr. Dimas Riveiro, Soledad Garcia, Dr. Samuel Ardvison, Dr. Martin Lauxmann, Romy Schmidt, Dr. Josefus Schippers, Claudia Sanabria, Justyna Tomala, Raul Trejos, Maryory Leyton, Niels Weisbach and Katharina Berger. All of them support me through my Ph.D taking me out of my routine, improving my research and making my life much happier and easier. I offer my most sincere gratitude towards Dr. Flavia Vischi, Dr. Fernando Arana, Dr. Diego Riaño-Pachón, Dr. Magarita Perea-Dallos and Dr. Nestor Riaño my close friends for their sincere support and because without your continue positivism and encourage it would have been impossible for me to begin and complete this work successfully. Special thanks to Family Brozio, Family Lieutenant, Family Peña-Andrade and Andrea Diaz for making me feel at home in Germany. The impulse to embark into this adventure and the encouragement and love to go to the end comes from my beloved friend and partner Hernando Leyton. He has never accepted less than my best efforts. Thank you. Lastly, I offer my deepest regards and blessings to my whole family especially to my beloved mom Cristina, my young brother Alejandro,