Influences of Gravitational Intensity on the Transcriptional Landscape of Arabidopsis thaliana A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Alexander D. Meyers May 2020 © 2020 Alexander D. Meyers. All Rights Reserved. 2 This dissertation titled Influences of Gravitational Intensity on the Transcriptional Landscape of Arabidopsis thaliana by ALEXANDER D. MEYERS has been approved for the Department of Molecular and Cellular Biology and the College of Arts and Sciences by Sarah E. Wyatt Professor of Environmental and Plant Biology Florenz Plassmann Dean, College of Arts and Sciences 3 Abstract MEYERS, ALEXANDER D, Ph.D., May 2020, Molecular and Cellular Biology Influences of Gravitational Intensity on the Transcriptional Landscape of Arabidopsis thaliana Director of Dissertation: Sarah E. Wyatt Plants use a myriad of environmental cues to inform their growth and development. The force of gravity has been a consistent abiotic input throughout plant evolution, and plants utilize gravity sensing mechanisms to maintain proper orientation and architecture. Despite thorough study, the specific mechanics behind plant gravity perception remain largely undefined or unproven. At the center of plant gravitropism are dense, specialized organelles called starch statoliths that sediment in the direction of gravity. Herein I describe a series of experiments in Arabidopsis that leveraged RNA sequencing to probe gravity response mechanisms in plants, utilizing reorientation in Earth’s 1g, fractional gravity environments aboard the International Space Station, and simulated fractional and hyper gravity environments within various specialized hardware. Seedlings were examined at organ-level resolution, and the statolith-deficient pgm-1 mutant was subjected to all treatments alongside wildtype seedlings in an effort to resolve the impact of starch statoliths on gravity response. In all, 132 unique genotype/tissue/treatment datasets were collected to help further our understanding of the gravitropic mechanisms in plants. 4 Dedication For Carl 5 Acknowledgments The work presented here represents a collaborative effort, and I would like to acknowledge the many people who made it possible. Thank you. 6 Table of Contents Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 4 Acknowledgments............................................................................................................... 5 List of Tables ...................................................................................................................... 8 List of Figures ..................................................................................................................... 9 Chapter 1: An Overview of Gravity Sensing and Response in Plants .............................. 15 Introduction ................................................................................................................. 15 Background ................................................................................................................. 15 Timeline of Gravity Response .................................................................................... 17 Gravity Sensing: The Role of Plastids ........................................................................ 18 Gravity Response: The Role of Auxin ........................................................................ 20 Other Players in Gravity Response ............................................................................. 23 Conclusions ................................................................................................................. 24 Chapter 2: Starch Statoliths and Their Role in Plant Gravity Response........................... 25 Introduction and History ............................................................................................. 25 Sensing Mechanisms ................................................................................................... 28 Evidence for Statoliths ................................................................................................ 31 Evolution of Starch Statoliths – Root Perspectives .................................................... 32 Statolith Production, Regulation, and Turnover ......................................................... 34 Starchless and Starch-Deficient Mutants .................................................................... 35 Cytoskeletal Features of the Statocyte ........................................................................ 36 Distinctions Between Root and Shoot Statocytes ....................................................... 37 Paradigms of Perception and Future Outlook ............................................................. 38 Chapter 3: Plant Gravity Perception – Molecular Definition for Spaceflight .................. 40 Introduction ................................................................................................................. 40 Post-Flight Definition ................................................................................................. 42 Plant Material, Sampling, and Replicates ................................................................... 43 Dissection .................................................................................................................... 47 Extraction .................................................................................................................... 50 Sequencing .................................................................................................................. 52 7 Validation .................................................................................................................... 53 Conclusions ................................................................................................................. 56 Chapter 4: Transcriptional Effects of Gravitational Reorientation in Starchless Mutants of Arabidopsis ....................................................................................................................... 57 Introduction ................................................................................................................. 57 Methods....................................................................................................................... 58 Plant Growth ......................................................................................................... 58 Treatment .............................................................................................................. 58 Processing ............................................................................................................. 59 Bioinformatics and Data Analysis ........................................................................ 60 Results ......................................................................................................................... 63 Contrast Analysis ........................................................................................................ 76 KEGG Pathway Enrichments - Reorientation ............................................................ 84 KEGG Pathway Enrichments - WT vs pgm-1 ............................................................ 96 Network Analysis...................................................................................................... 114 Discussion ................................................................................................................. 118 Root Tip Differential Expression .............................................................................. 118 Hypocotyl Differential Expression ........................................................................... 122 Cotyledon Differential Expression ........................................................................... 124 Mature Root Differential Expression ........................................................................ 124 Metabolism in pgm-1 ................................................................................................ 126 Contrast Analyses ..................................................................................................... 127 Conclusions ............................................................................................................... 130 Chapter 5: Future directions............................................................................................ 132 PGP Flight ................................................................................................................. 134 PGP-ESTEC .............................................................................................................. 135 Results, Conclusions, & Future Directions ............................................................... 137 References ....................................................................................................................... 143 Appendix: Methods for Plant Growth and RNA Extraction for Polyethersulfone (PES) Membranes ...................................................................................................................... 152 8 List of Tables Page