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IRON HOMEOSTASIS IS HIERARCHICALLY REGULATED BY MULTIPLE INPUTS: EVIDENCE FOR THE ROLE OF REACTIVE OXYGEN SPECIES AND IRON-ZINC CROSS TALK __________________________________________ A Dissertation Presented to The Faculty of the Graduate School at the University of Columbia - Missouri ____________________________________ In partial fulfillment of the Requirements for the Degree Doctor of Philosophy ______________________________ By SAMUEL ALONZO MCINTURF ________________________ Dr. Mendoza-Cózatl, Dr. Gassmann, Dr. Hunt, Dr. Kazic, Dr. Mitchum Dissertation Supervisors December 2018 The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled IRON HOMEOSTASIS IS HEIRARCHICALLY REGULATED BY MULTIPLE INPUTS: EVIDENCE FOR THE ROLE OF REACTIVE OXYGEN SPECIES AND IRON-ZINC CROSS TALK Presented by Samuel Alonzo McInturf a candidate for the Degree of Doctor of Philosophy, and herby certify that, in their opinion, is worthy of acceptance. ______________________________________________________ Professor David Mendoza-Cózatl ______________________________________________________ Professor Walter Gassmann ______________________________________________________ Professor Heather Hunt ______________________________________________________ Professor Toni Kazic ______________________________________________________ Professor Melissa Mitchum William Alonzo McInturf For my father who has always demonstrated a dedication to hard, honest work. Lanelle Jane McInturf For my mother, who has been continually supportive, through all of my phases. Dr. Molly Alona McCarthy For my sister, who has shown me what it means to persevere and flourish in face of attrition and hardship. To these people I owe my circumstance which has allowed me to come so far. ACKNOWLEDGEMENTS I would like to thank Dr. Mendoza-Cózatl for the years of support, for the challenges brought, and met, and those lost as well. His insights and guidance have led me to grow both as a scientist and as a person, more than I thought I ever would. For this trial, for this burden, for this growth, and for this joy, I am deeply grateful. I would like to thank Dr. Kazic for first showing me, and then reminding me why computational biology is beautiful and for helping me through the hardships. I would like to thank Dr. Mitchum and Dr. Gassmann for overseeing my growth and for the advice and lessons they passed down. I would like to thank Dr. Hunt showing me how to manage groups, for her advice, but most importantly for reminding me where my feet stand, and that the sky resides above that point. For the Doctors’ Dowd, for their years of friendship, without which I dare not consider. My lab mates, Dr. Mather Khan who brought me half way around the world. For Dr. Nga Ngyuen, for the comradery of the journey. For Dr. Norma Castro-Guerrero for reminding me that, we just don’t think about “The Spaghetti Incident?”. For my friends with whom I counted the small hours, and for those with whom I have spun my most favored yarns. Ph'nglui mglw'nafh Cthulhu R'lyeh wgah'nagl fhtagn! ii TABLE OF CONTENTS Acknowledgements.......................................................................................................................... ii Table of Figures ............................................................................................................................. viii Table of Supplemental Figures ........................................................................................................ x Abstract ............................................................................................................................................ x Chapter 1 Iron Homeostasis 1 Introduction ............................................................................................................................. 1 2 The Fe uptake pathway ............................................................................................................ 3 2.1 The reduction strategy for Fe2+ uptake ............................................................................ 4 2.2 Regulation of reduction strategy uptake by the FIT network .......................................... 5 2.3 Fe mobilization from the rhizosphere ............................................................................. 7 3 The Chelation strategy of Fe uptake (Strategy II) .................................................................... 9 4 The PYE network regulates intracellular Fe homeostasis in leaves and roots....................... 11 4.1 Fe-citrate complexes are translocated from roots to leaves via the xylem .................. 15 5 Local regulation of Fe deficiency ........................................................................................... 16 6 The systemic Fe deficiency signal and the role of companion cells in leaves........................ 18 6.1 Fe homeostasis in photosynthetically active cells is maintained by the chloroplast- vacuole shuttle ........................................................................................................................... 20 6.2 The systemic Fe sensor .................................................................................................. 21 7 Conclusion .............................................................................................................................. 25 8 Bibliography ........................................................................................................................... 27 Chapter 2 Cadmium interference with iron sensing reveals transcriptional programs sensitive and insensitive to reactive oxygen species 1 Abstract .................................................................................................................................. 34 2 Introduction ........................................................................................................................... 35 3 Results .................................................................................................................................... 39 3.1 Mild Cd exposure induces a partial Fe deficiency response in roots and leaves. .......... 39 iii 3.2 Fe overload partially restricts the Cd-induced Fe deficiency response. ........................ 42 3.3 Fe deficiency responses are hierarchically regulated based on the levels of reactive oxygen species. .......................................................................................................................... 46 3.4 H2O2 over accumulates in leaves and roots of opt3-2 ................................................... 49 3.5 Inhibition of photosynthesis by Cd is a source for the elevated levels of H2O2. ............ 50 4 Discussion............................................................................................................................... 52 4.1 Cd induces Fe deficiency by impairing Fe sensing ......................................................... 53 4.2 Fe deficiency is hierarchically regulated by competing nutrient acquisition and oxidative stress signals. ............................................................................................................................. 55 5 Conclusion .............................................................................................................................. 56 6 Materials and Methods .......................................................................................................... 57 6.1 Plant growth ................................................................................................................... 57 6.2 RNA sequencing and data analysis ................................................................................ 57 6.3 Hydrogen peroxide quantification ................................................................................. 58 7 Bibliography ........................................................................................................................... 59 Chapter 3 bZIP23 as a putative link between zinc and iron homeostasis 1 Abstract .................................................................................................................................. 65 2 Introduction ........................................................................................................................... 66 3 Results .................................................................................................................................... 70 3.1 bzip23-1/opt3-2, but not bzip23-2/opt3-3, is able to completely suppress opt3-2 phenotypes ................................................................................................................................ 71 3.2 The o2b231 mutant contains wild type levels of heavy metals ...................................... 76 3.3 mRNA profiling demonstrates the extent of opt3 suppression in o2b231 ......................... 77 3.4 bZIP23 overexpression complements the o2b231 phenotype and suggests post- translational regulation of bZIP23. ............................................................................................ 80 3.5 bZIP23 localizes to the vasculature of leaves and roots ................................................ 82 3.6 bZIP23 binds to the promoters of the Fe uptake pathway in a protein-DNA binding screens (Y1H) ............................................................................................................................. 84 3.7 DAFL1 as a putative regulator of bZIP23.......................................................................

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