Western University Scholarship@Western Electronic Thesis and Dissertation Repository 11-3-2017 10:00 AM Characterization of six Arabidopsis AROGENATE DEHYDRATASE promoters Emily J. Cornelius The University of Western Ontario Supervisor Dr. Susanne E. Kohalmi The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Emily J. Cornelius 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biology Commons Recommended Citation Cornelius, Emily J., "Characterization of six Arabidopsis AROGENATE DEHYDRATASE promoters" (2017). Electronic Thesis and Dissertation Repository. 5081. https://ir.lib.uwo.ca/etd/5081 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Phenylalanine is an important aromatic amino acid synthesized by higher plants, and is a major component of numerous specialized metabolites including structural components, pigments, and defense compounds. The last step in the synthesis of phenylalanine is catalyzed by an enzyme called AROGENATE DEHYDRATASE, of which there are six different isoenzymes encoded by the Arabidopsis genome. All six have specialized roles within the plant, and are differentially expressed during development and under stressful conditions. To deduce the potential specialized role of each ADT, unique patterns of regulatory motifs were identified for all six ADT promoters, as well as corresponding transcription factors with similar expression profiles to each enzyme. Seven stable transgenic Arabidopsis lines were also generated using ADT promoter-eGFP/GUS constructs to test expression in all tissues during development, and under stressful conditions. Keywords Phenylalanine, specialized metabolism, arogenate dehydratase, cis regulatory motif, co-expression, transcriptional regulation i Acknowledgments Firstly, I would like to thank my supervisor, Dr. Susanne Kohalmi, for her undying guidance, support, and mentorship throughout this thesis. Thank you for giving me the chance to learn, and for teaching me countless lessons about science, research, and life. I will be forever grateful for the opportunities you have given me along this entire journey. I would also like to thank my advisors, Dr. Norm Huner and Dr. Ryan Austin, for their much appreciated support and contributions throughout the process. Second, I would like to thank Emily Clayton, Sara Rad, and Caryn Dooner, not only for being the most wonderful lab mates, but also great friends. Your tireless support and encouragement means the world to me. Also thanks to Jessica Belcastro, Daniel Bour and Michelle Millones. Daily life in the lab would not have been the same without all of you. Third, I would like to thank my friends inside and outside the department, especially Tian Wu. Thank you for helping me realize my potential, and for keeping a smile on my face throughout every challenge. Thank you also to the rest of my friends for all the memories that I am so grateful to take with me from the last two years. I will truly miss watching ever major television event together. Finally, I would like to thank my family. Your unconditional love and support gives me the courage to persevere when times are at their most difficult. ii Table of Contents Abstract ........................................................................................................... i Acknowledgments ......................................................................................... ii List of Tables……………………………………………………………………...vii List of Figures .............................................................................................. vii List of Appendices ..................................................................................... viii List of Abbreviations .................................................................................... ix 1 INTRODUCTION .......................................................................................... 1 1.1 Secondary Metabolism and the Phenylpropanoid Pathway ...................... 1 1.2 Phenylalanine Biosynthesis ....................................................................... 3 1.3 Arogenate Dehydratases ........................................................................... 4 1.3.1 Alternative Roles and Differential Expression ........................................ 7 1.4 Transcriptional Regulation in Eukaryotes .................................................. 8 1.5 Promoter Organization ............................................................................ 14 1.6 Duplications and Gene Family Evolution ................................................. 18 1.7 A Data Mining Approach to Gene Family Analysis .................................. 21 1.8 Research Questions, Experimental Design and Objectives .................... 23 2 MATERIALS AND METHODS ................................................................... 24 2.1 Media, Solutions and Buffers .................................................................. 24 2.1.1 Media .................................................................................................... 24 2.1.2 Solutions ............................................................................................... 24 2.1.3 Buffers .................................................................................................. 25 2.2 Bacterial Strains and Plasmids ................................................................ 25 2.2.1 Bacterial Strains and Growth Conditions .............................................. 25 2.2.2 Plasmids ............................................................................................... 26 2.3 Plant Material and Standard Growth Conditions ..................................... 26 2.4 DNA Isolation .......................................................................................... 28 2.4.1 Plasmid DNA Isolation from Bacteria ................................................... 28 2.4.2 Plant Genomic DNA Isolation ............................................................... 28 2.5 PCR Amplification and Purification of ADT Promoter Regions ............... 28 iii 2.5.1 Primer Design and Sequence Amplification ......................................... 30 2.5.2 Gel Electrophoresis of PCR Products and DNA Extraction .................. 30 2.6 Gateway Cloning Procedure ................................................................... 30 2.7 Transformations ...................................................................................... 31 2.7.1 E. coli Transformations ......................................................................... 31 2.7.2 A. tumefaciens Transformations ........................................................... 31 2.7.3 N. benthamiana Transformations ......................................................... 34 2.7.4 A. thaliana Transformations ................................................................. 34 2.8 Seed Collection, Sterilization and Storage .............................................. 35 2.9 Histochemical Detection of GUS ............................................................. 35 2.10 Confocal Microscopy ............................................................................. 35 2.11 In Silico Methods ................................................................................... 36 2.11.1 Sequence Analyses ............................................................................ 36 2.11.2 Motif Pattern Analysis ......................................................................... 36 2.11.3 Co-expression Analysis ...................................................................... 37 3 RESULTS ................................................................................................... 38 3.1 In Silico Results ....................................................................................... 38 3.1.1 Sequence Analyses .............................................................................. 38 3.1.2 Regulatory Motif Categories ................................................................. 41 3.1.3 Proportions of Motif Categories in Each Promoter ............................... 45 3.1.4 Motifs Common to all Promoters .......................................................... 45 3.1.5 Unique Motifs ....................................................................................... 51 3.1.6 Significantly Enriched Motifs ................................................................ 58 3.1.7 Co-expression Results ......................................................................... 67 3.2 Cloning and Expression of ADT Promoter-Reporter Constructs. ............ 76 3.2.1 Promoter Sequence Amplification from gDNA ..................................... 79 3.2.2 Construct Confirmation ......................................................................... 80 3.2.3 Transient Expression by ADT Promoter Sequences ............................ 80 3.2.4 Stable Transformations of Arabidopsis ................................................ 89 4 DISCUSSION ............................................................................................. 92 4.1 ADT Regulation and Alternative Roles ...................................................