UC Riverside UC Riverside Electronic Theses and Dissertations Title Regulatory Mechanisms of Low Oxygen Sensing and Response in Arabidopsis thaliana Permalink https://escholarship.org/uc/item/2g75p3nv Author Lee, Seung Cho Publication Date 2012 Supplemental Material https://escholarship.org/uc/item/2g75p3nv#supplemental Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Regulatory Mechanisms of Low Oxygen Sensing and Response in Arabidopsis thaliana A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Biology by Seung Cho Lee September 2012 Dissertation Committee: Dr. Julia Bailey-Serres, Chairperson Dr. Xuemei Chen Dr. Harley Smith i Copyright by Seung Cho Lee 2012 ii The Dissertation of Seung Cho Lee is approved: _______________________________________________ _______________________________________________ _______________________________________________ Committee Chairperson University of California, Riverside iii Acknowledgments I would like to thank my advisor, Prof. Julia Bailey-Serres for her support and guidance throughout my PhD program. I was fortunate to conduct research under her guidance. Because of my PhD projects, my knowledge of plant molecular biology has grown immensely. I also thank Prof. Xuemei Chen and Prof. Harley Smith for valuable comments on my research as members of my dissertation committee. I am also grateful to the current and former members of the Bailey-Serres lab throughout my 5 years here for their discussion, assistance, and friendship. I would especially like to thank Dr. Takeshi Fukao, Prof. Angelika Mustroph, Dr. Teruko Oosumi, Dr. Julian Pena-Castro, Dr. Piyada Juntawong, Reed Sorenson, Charles Jang, Erin Brinton, Maureen Hummel, Kayla Hamersky Kaiser, and Greg Barding. Also, many thanks to my undergraduate researchers, Julian Kougl and Jonathan Dang, for their assistance with my projects. I would like to thank all my project collaborators including Prof. Thomas Girke for helping with the bioinformatic analyses, Prof. Rens Voesenek and Divya Vashisht at Utrecht University for assistance and suggestions for the submergence experiments, Prof. Mike Holdsworth and Dr. Daniel Gibbs at the University of Nottingham for their collaboration and assistance with the N-end rule project, and Prof. Pierdomenico Perata and Dr. Beatrice Giuntoli at Scuola Superiore Sant'Anna for collaboration on the HRA1 project. Lastly, I would like to thank my parents and my wife Chanmi Yang. I could not have finished my PhD successfully without their warm support and prayers. iv Publications Mustroph A, Lee SC, Oosumi T, Zanetti ME, Yang H, Ma K, Yaghoubi-Masihi A, Fukao T, Bailey-Serres J. 2010. Cross-kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses. Plant Physiology, 152:1484-500. Lee SC, Mustroph A, Sasidharan R, Vashisht D, Pedersen O, Oosumi T, Voesenek LA, Bailey-Serres J. 2011. Molecular characterization of the submergence response of the Arabidopsis thaliana ecotype Columbia. New Phytologist, 190:457-471. (Modified to conform to dissertation format). Gibbs DJ, Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J, Holdsworth MJ. 2011. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature, 479: 415- 418. v ABSTRACT OF THE DISSERTATION Regulatory Mechanisms of Low Oxygen Sensing and Response in Arabidopsis thaliana by Seung Cho Lee Doctor of Philosophy, Graduate Program in Plant Biology University of California, Riverside, September 2012 Dr. Julia Bailey-Serres, Chairperson Flooding is a major natural disaster that is responsible for significant crop loss worldwide. Ethylene accumulation and reduced oxygen availability are typical changes upon submergence in plant tissues. Researchers identified two antithetical strategies, ‘escape’ and ‘quiescence’, for survival of submergence. These are manifested in rice (Oryza sativa) through regulation of growth and carbohydrate consumption by group VII ETHYLENE RESPONSIVE FACTOR (ERF) transcription factors. The low oxygen response of Arabidopsis thaliana, a model dicot species, includes transcriptional regulation. Independent transcriptomic analyses using microarray technology identified about 50 core hypoxia-responsive genes, including over 25 encoding proteins of unknown function. In this study, in planta measurement of oxygen content revealed distinct dynamics in root and shoot tissues upon submergence. Microarray analysis was conducted to dissect the molecular response to submergence of these organs in rosettes and for comparison to that of roots and shoots of hypoxic seedlings. Studies of loss-of- function and gain-of-function mutants of HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes identified conserved genes that contribute to low oxygen and vi submergence tolerance in Arabidopsis. Of these, a previously uncharacterized protein was recognized as a negative transcriptional regulator of the hypoxia response. This protein, HYPOXIA RESPONSIVE ATTENUATOR1 (HRA1), contains a trihelix domain and functions as a transcriptional activator in yeast. Overexpression of HRA1 dampened transcription of core hypoxia-responsive genes in seedlings. Investigation of N-end rule pathway of targeted proteolysis (NERP) unveiled a mechanism of direct sensing of oxygen availability in Arabidopsis. Mutation of genes encoding NERP components resulted in constitutive expression of many core hypoxia-responsive genes. Five Arabidopsis group VII ERFs are positive regulators of transcription in response to hypoxia. One of these was shown to be unstable in seedlings maintained in air and stabilized upon transfer to low oxygen condition and in mutants lacking NERP activity. This and the observation that the mutation of the second amino acid of these proteins from cysteine to alanine, provide strong evidence that Arabidopsis group VII ERFs are substrates of NERP in an oxygen-dependent manner. This dissertation provides new insights into molecular mechanisms of low oxygen sensing and response in Arabidopsis, which may be conserved in other plant species. vii Contents Page List of tables .....................................................................................................................xv List of figures ................................................................................................................. xvi List of supplementary tables ............................................................................................xx 1. Sensing, signaling and response to low oxygen in plants 1.1. Introduction ...............................................................................................................1 1.2. Features and responses at organ and tissue-levels that contribute to flooding survival ................................................................................................4 1.2.1. Shoot elongation in rice, Rumex species and Arabidopsis under submergence ......................................................................................4 1.2.2. Developmental adaptation to flooding in plants ..........................................7 1.3. Regulation of gene expression and metabolism in response to low oxygen in plants ........................................................................................10 1.3.1. Common and unique hypoxia-induced genes among different cell-types and low oxygen regimes ............................................................10 1.3.2. Regulation of gene transcription during hypoxia .......................................12 1.3.3. Translational and epigenetic regulation of the low oxygen response ........................................................................15 1.3.4. Anaerobic metabolism ................................................................................17 1.3.5. Signaling by energy sensing (SnRK1, CIPK, KIN10, KIN11) and stress responses ...................................................................................19 viii 1.4. Low oxygen sensing and signaling in plants ........................................................21 1.4.1. Indirect sensing by energy, calcium, ROS, and G proteins ........................21 1.4.2. Direct oxygen sensing ..................................................................................23 1.5. Dissertation objectives ..........................................................................................24 2. Initial functional characterization of HYPOXIA-RESPONSIVE UNKNOWN PROTEINs of Arabidopsis thaliana 2.1. Abstract .................................................................................................................41 2.2 Introduction ............................................................................................................42 2.3. Materials and methods ..........................................................................................45 2.3.1. Microarray data used in the analysis ..........................................................45 2.3.2. Phenotypic analysis of HUP mutants under prolonged low oxygen stress ............................................................46 2.4. Results ...................................................................................................................48 2.4.1. Molecular characteristics of HUPs ............................................................48