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Transcriptional and Post-Translational Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus Roseus

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Transcriptional and Post-Translational Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus Roseus University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2017 TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN CATHARANTHUS ROSEUS Priyanka Paul University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0001-8530-7877 Digital Object Identifier: https://doi.org/10.13023/ETD.2017.403 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Paul, Priyanka, "TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN CATHARANTHUS ROSEUS" (2017). Theses and Dissertations--Plant and Soil Sciences. 94. https://uknowledge.uky.edu/pss_etds/94 This Doctoral Dissertation is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Priyanka Paul, Student Dr. Ling Yuan, Major Professor Dr. Mark Coyne, Director of Graduate Studies TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN CATHARANTHUS ROSEUS ____________________________________________________ DISSERTATION ____________________________________________________ A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By Priyanka Paul Director: Dr. Ling Yuan, Professor of Department of Plant and Soil Sciences Lexington, Kentucky 2017 Copyright © Priyanka Paul 2017 ABSTRACT OF DISSERTATION TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN CATHARANTHUS ROSEUS Catharanthus roseus (Madagascar periwinkle) is the exclusive source of an array of terpenoid indole alkaloids (TIAs) that are used in the treatments of hypertension and certain types of cancer. TIA biosynthesis is under stringent spatiotemporal control and is induced by jasmonate (JA) and fungal elicitors. Tryptamine, derived from the indole branch, and secologanin from the iridoid branch are condensed to form the first TIA, strictosidine. Biosynthesis of TIA is regulated at the transcriptional level and several transcription factors (TFs) regulating the expression of genes encoding key enzymes in the pathway have been isolated and characterized. The JA-responsive APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3, and the basic helix-loop-helix (bHLH) factor, CrMYC2, are the key activators of the TIA biosynthesis. Recently, two other TFs, the bHLH IRIDOID SYNTHESIS 1 (BIS1) and BIS2 were also identified as regulators of TIA pathway. Analysis of C. roseus genome sequence has revealed that ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and ORCA5. In plants, physically linked clusters of TFs are less characterized. Moreover, the regulation of TF clusters is relatively unexplored. My research uncovered that the ORCA gene cluster is differentially regulated. ORCA4 and ORCA5, while functionally overlapping with ORCA3, regulate an additional set of TIA pathway genes. ORCA4 or ORCA5 overexpression has resulted in significant increase of TIA accumulation in C. roseus hairy roots. In addition, ORCA5 directly regulates the expression of ORCA4 and indirectly regulates ORCA3, likely via unknown factor(s). Interestingly, ORCA5 also activates the expression of ZCT3, a negative regulator of the TIA pathway. In addition CrMYC2 is capable of activating ORCA3 and co-regulating pathway genes concomitantly with ORCA3. Several lines of evidence suggest that, in addition to the transcriptional control, biosynthesis of TIAs is also controlled at the posttranslational level, such as protein phosphorylation. Available literature indicates that a mitogen-activated protein kinase (MAPK) cascade is involved in this process. Analysis of C. roseus MAP kinome, identified two independent MAPK cascades regulating the indole and iridoid branches of the TIA pathway. We showed that the ORCA cluster and CrMYC2 act downstream of a MAP kinase cascade consisting of CrMAPKK1, CrMAPK3 and CrMAPK6. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulates TIA pathway genes expressions and boosts TIA accumulation. The other cascade, consisting of CrMAPKK6 and CrMAPK13, mostly regulates the iridoid branch of the TIA pathway. Overexpression of CrMAPK13 in C. roseus hairy roots significantly upregulates iridoid pathway genes and boosts tabersonine accumulation. Moreover, we recently identified the third MAPK cascade, consisting of CrMAPKK1 and CrMAPK20, that negatively regulates the indole branch of the TIA pathway. Overexpression of CrMAPK20 in C. roseus hairy roots represses the genes regulated by CrMYC2-ORCAs and reduces catharanthine accumulation. These findings significantly advance our understanding of transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus. KEY WORDS: AP2/ERF gene cluster, Catharanthus roseus, MAP kinome, terpenoid indole alkaloids, transcriptional and post-translational regulation. Priyanka Paul 08-28-17 TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN CATHARANTHUS ROSEUS By Priyanka Paul Dr. Ling Yuan Director of Dissertation Dr. Mark Coyne Director of Graduate Studies 08-28-17 To my beloved husband Sanjay A man with love and support ACKNOWLEDGMENTS I am grateful to so many people for their love and support over the past years. I would first like to express my sincere gratitude to my advisor Prof. Ling Yuan for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D. study. Besides my advisor, I would like to thank the rest of my Ph.D. committee: Prof. Arthur Hunt, Dr. Sharyn Perry, and Dr. Xuguo Zhou not only for their insightful comments and encouragement, but also for the hard questions which gave me incentive me to widen my research and expand my perspectives. I would also like to thank Prof. Subba Reddy Palli who served as my outside examiner. The members of Prof. Yuan lab are a great group of people and I have enjoyed working with them for the past several years. It is just like my second home. I would like to thank Dr. Sitakanta Pattanaik who, with great patience in every situation, trained me in my research and with his great kindness he is always with me in every situation. I thank him for correcting and proofreading this dissertation. Without his precious support it would not have been possible to conduct this research. I also thank Kathy Shen who proofread and corrected the language of my research papers. I sincerely thank Dr. Baruvana Patra and Dr. Sanjay Singh for their great help in conducting my research. The many discussions with Dr. Baruvana Patra, Dr. Sanjay Kumar Singh, Dr. Jayadri Shekhar Ghosh and Dr. Yongliang Liu were not only helpful but provided much encouragement. I sincerely thank Dr. Craig Schluttenhofer for proofreading this dissertation. I thank all my lab mates and friends, Dr. Craig Schluttenhofer and Dr. Xueyi Sui for the stimulating discussions and for all the fun we have had in the last four years. I am also iii grateful to the visiting scholar Guiping Ren, Xiaoyu Liu and former lab members Dr. Yongmei Wu, Dr. Chunmei Zhong, Dr. Tiandai Huang, Dr. Shiyuan Deng and Dr. Yuxiang Wu for a congenial environment. Additionally, many other people from the KTRDC and UK Plant and Soil Sciences Department also deserve thanks, ranging from other graduate students to postdocs to technicians and professors. My dream of doing a Ph.D. would not have been possible without the constant love, support, and encouragement from my life partner
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