The Integration of Metabolite and Hormone Signalling Drives Seedling Development by Michael Stokes 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 Michael Stokes ii The Integration of Metabolite and Hormone Signalling Drives Seedling Development Michael Stokes Doctor of Philosophy Department of Cell & Systems Biology, University of Toronto 2013 Abstract Sugars have a profound impact on plant biology, acting as structural components, signalling molecules, and sources of energy. As such, the availability of sugars has important implications for plant growth and development. Sugar levels rise and fall as part of a daily cycle, while cues from other environmental stimuli are also in flux. As sessile organisms in an ever-changing environment, plants must integrate signals from multiple pathways in order to promote the appropriate developmental responses. To uncover pathways that interact with sugars during seedling development, a chemical screen was performed in search of compounds that modify responses to sucrose. This screen identified an interaction between the folate inhibitor sulfamethoxazole (SMX) and sucrose that resulted in changes to auxin signalling and distribution. Synergy between sucrose and SMX was used to explore the effect of metabolic cues on iii auxin signalling during hypocotyl elongation. A second line of investigation explored whether sucrose and folates influence root meristem activity. Sucrose induced hormone signalling pathways that promote cell division and differentiation. Treatment with SMX perturbed the effect of sucrose on hormone networks that mediate growth, and resulted in a loss of meristem integrity. This study highlights the influence of metabolism on hormone signalling at the root apex, and its role in maintaining balance of a complex signalling network that drives root growth. These studies characterise an interaction between metabolic pathways that is integrated with hormone signalling during plant development. Taken together, they highlight a mechanism through which plant growth might be regulated by metabolism. iv Acknowledgements Firstly, I would like to thank my supervisor, Dr Malcolm Campbell, for his generous support and guidance throughout the time I have spent in his lab. He has been a great mentor, and I am grateful for having had to the opportunity to work with his group. I would also like to thank Joan Ouellette, who has put in countless hours keeping the lab running smoothly. Without her support over the years, this thesis would not have been possible. I would also like to thank the members of my Advisory Committee, Dr Darrell Desveaux and Dr Peter McCourt, for guiding my research efforts, their insight and thoughtful advice is truly appreciated. Thank you to Dr Eiji Nambara and Dr Philip Benfey, who have devoted their valuable time to review my thesis and take part in my final defence. I would like to acknowledge the guidance and friendship of my labmates, who have helped with brainstorming new ideas, and then with troubleshooting. It has been great working with you, especially Erin Hamanishi, Mike Prouse, Rohan Patel, Heather Wheeler, and Joseph Skaf. Special thanks to the graduate students and postdocs in other labs that have taken time to help me with protocols or to offer advice, especially Dr Steven Chatfield, Dr Amy Lee, Dr Karl Schreiber and Dr Wenzi Ckurshumova. I would like to thank my parents Kevin and Rose, and my sisters Adrienne and Jacqueline, for their encouragement, love and support. Without them I would not be here. I would like to thank my friends, especially Aarie Glas and Evan Mackenzie, who offered comic relief at the times it was needed. Above all, I would like to thank Charlotte Burke for her warmth, kindness, and understanding. Her love has made all the difference. v Table of Contents ACKNOWLEDGEMENTS ........................................................................... V LIST OF ABBREVIATIONS ....................................................................... IX LIST OF FIGURES .................................................................................. XIII LIST OF TABLES ..................................................................................... XV CHAPTER 1: INTRODUCTION .................................................................. 1 1.1 Sugars have a profound impact on plant development ...................................... 2 1.2 Sugar levels rise and fall throughout the day, with widespread consequences .................................................................................................... 3 1.3 Sugars are perceived, resulting in changes to gene expression ........................ 5 1.4 Transcriptome analysis to uncover pathway interactions ................................... 7 1.5 Sugars and hormones ........................................................................................ 7 1.6 Sugars and auxin coordinate plant development ............................................... 8 1.7 Sugars impact the plant metabolome; metabolites can act as signalling.............. molecules that regulate gene expression ........................................................ 12 1.8 Folates effect plant development and productivity ........................................... 12 1.9 Chemical inhibition of THF biosynthesis as a tool for plant sciences ............... 16 1.10 Overcoming redundancy: chemical genetics .................................................... 18 1.11 Combination chemical genetics ........................................................................ 21 1.12 RESEARCH QUESTIONS AND HYPOTHESESError! Bookmark not defined.22 CHAPTER 2: INTERPLAY BETWEEN SUCROSE AND FOLATE MODULATES AUXIN SIGNALLING IN ARABIDOPSIS. .......................... 25 2.1 ABSTRACT ...................................................................................................... 25 2.2 INTRODUCTION .............................................................................................. 26 vi 2.3 RESULTS ......................................................................................................... 28 2.3.1 Sulfonamides alter seedling responses to sucrose ................................... 28 2.3.2 Sulfamethoxazole impinges on sucrose-mediated development through inhibition of Dihydropteroate Synthase (DHPS) ........................................ 29 2.3.3 Changes in transcript abundance of specific auxin signal transduction components occurs in response to the combined action of SMX and sucrose ...................................................................................................... 34 2.3.4 Interaction between sucrose and SMX is dependent on auxin transport and signal transduction .................................................................................... 40 2.3.5 Sucrose and SMX promote auxin signalling in vasculature tissues of the hypocotyl ................................................................................................... 44 2.4 DISCUSSION ................................................................................................... 45 2.5 MATERIALS AND METHODS ......................................................................... 52 2.5.1 Plant material and growth conditions ......................................................... 52 2.5.2 Chemical screen........................................................................................ 52 2.5.3 Dose response curves and chemical treatments ....................................... 53 2.5.4 Tissue preparation, RNA extraction, and microarray analysis ................... 53 2.5.5 Quantitative RT-PCR ................................................................................. 55 2.5.6 Creation of GUS reporter constructs and histochemical analysis .............. 55 2.5.7 Confocal microscopy ................................................................................. 56 2.5.8 Auxin quantification ................................................................................... 57 2.6 ACKNOWLEDGEMENTS ................................................................................ 58 2.7 SUPPLEMENTAL MATERIAL .......................................................................... 59 CHAPTER 3: METABOLITE AND HORMONE SIGNALLING IS INTEGRATED AT THE MERISTEM TO DRIVE ROOT GROWTH ........... 75 3.1 ABSTRACT ...................................................................................................... 75 3.2 INTRODUCTION .............................................................................................. 76 3.3 RESULTS ......................................................................................................... 80 3.3.1 Sulfamethoxazole inhibits the effect of sucrose on root elongation ........... 80 vii 3.3.2 Sucrose promotes auxin accumulation at the root tip, which is inhibited by folate deprivation ....................................................................................... 80 3.3.3 PIN expression may underpin changes in auxin distribution caused by sucrose and SMX ...................................................................................... 82 3.3.4 Folate deprivation causes ectopic cytokinin signalling .............................. 84 3.3.5 Folates are necessary for cell cycle progression in response to sucrose .. 88 3.3.6 Meristem development is promoted by sucrose and depndent on folates . 90 3.3.7 Phenotypic