Transcriptional Control of PIN Auxin Efflux Carriers by Cytokinin

Ghent University Faculty of Sciences Department of Plant Biotechnology and Genetics Transcriptional control of PIN auxin efflux carriers by cytokinin Mária Šimášková Promoter: Prof. Dr. Eva Benková VIB / Plant Systems Biology Technologiepark 927, B-9000 Ghent, Belgium This thesis is submitted as fulfilment of the requirements for the degree of PhD in Sciences, Biotechnology Research presented in this thesis was performed at the University of Ghent, VIB, Department of Plant Systems Biology. “Meet the true lords of life, the plants. Life could get along without animals and without fungi. But abolish the plants, and life would rapidly cease.” - Richard Dawkins Promoter: Prof. Dr. Eva Benková VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Institute of Science and Technology Austria (IST Austria) Am Campus 1,A – 3400 Klosterneuburg AUSTRIA Email: [email protected] Examination committee: Prof. Dr. Tom Beeckman (chair) VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Prof. Dr. Dolf Weijers* Wageningen University Laboratory of Biochemistry Dreijenlaan 3, 6703 HA Wageningen The Netherlands Email: [email protected] Prof. Dr. Sofie Goormachtig * VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Dr. Ive De Smet* VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Dr. Steffen Vanneste* VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Prof. Dr. Dominique Van Der Straeten Faculty of Sciences, Ghent University K.L. Ledeganckstraat 35 B-9000 Ghent, Belgium [email protected] Prof. Dr. Frank Van Breusegem VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Dr. Moritz Nowack VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] Dr. Boris Parizot VIB / Ghent University, Department of Plant Systems Biology Technologiepark 927 B - 9052 Gent BELGIUM Email: [email protected] * reading committee TABLE OF CONTENTS Frequently used abbreviations ................................................................................................ 2 Scope .......................................................................................................................................... 4 Chapter 1. ................................................................................................................................... 7 Introduction ................................................................................................................................ 7 Plant hormones shaping the plant body .................................................................................. 9 1 Auxin ............................................................................................................................. 10 1.1 Auxin biosynthesis .................................................................................................. 10 1.2 Auxin transport ....................................................................................................... 12 1.3 Auxin transport regulation ..................................................................................... 15 1.4 Auxin perception and signaling .............................................................................. 16 1.5 Auxin as key player in plant development .............................................................. 18 1.6 Auxin as a trigger of embryogenesis ...................................................................... 19 1.7 Auxin and postembryonic development of lateral organs ...................................... 21 2 Cytokinins ..................................................................................................................... 25 2.1 Cytokinin metabolism ............................................................................................. 25 2.2 Transport of cytokinin ............................................................................................ 27 2.3.1 Signal sensing via histidine kinases ................................................................ 30 2.3.2 Histidine phosphotransferases act as cytoplasmic nuclear shuttles ................ 31 2.3.3 Mediating the transcriptional response to CK via RRs ................................... 32 2.3.4 New components of CK responses .................................................................. 35 3 Role of CK in plant development underlined by crosstalk with auxin ......................... 36 3.1 CK signaling and crosstalk with auxin in early embryogenesis ............................ 37 3.2 The negative role of CK in root development is counteracted by auxin ................ 38 3.3 CK controls shoot development in a positive fashion via interaction with a local transcriptional network and auxin ............................................................................... 41 References ........................................................................................................................... 45 Chapter 2. ................................................................................................................................. 63 Cytokinin regulates root meristem activity via modulation of the polar auxin transport ........ 63 Abstract ................................................................................................................................ 65 Introduction .......................................................................................................................... 66 Results and Discussion ......................................................................................................... 67 The CK Effect on the Root Meristem Does Not Interfere with Ethylene- Mediated Processes .......................................................................................................................... 67 CK and Nontransportable Auxin Show Similar Effects on the Root Meristem ................ 69 CK Reduces Auxin Efflux in Tobacco BY-2 Suspension Cells ......................................... 71 CK Modulates Expression of the Auxin Efflux Carriers of the PIN Family .................... 72 CK Effect on PIN1 Transcription Requires Histidine Kinase-Based CK Signaling ........ 73 Model for Auxin–CK Interaction in Root Meristems ....................................................... 75 Materials and Methods ......................................................................................................... 77 Plant Materials and Growth Conditions .......................................................................... 77 PIN1::mRFP1 Expression Analysis in BY-2 Cells .......................................................... 78 Auxin Accumulation Assays ............................................................................................. 78 Quantitative RT-PCR ....................................................................................................... 78 Acknowledgements .............................................................................................................. 79 Supplementary Figures ......................................................................................................... 80 References ............................................................................................................................ 85 Chapter 3. ................................................................................................................................. 89 The Cytokinin Response Factors regulate PIN gene transcription .......................................... 89 Abstract ............................................................................................................................... 91 Introduction ........................................................................................................................ 92 Results and Discussion ....................................................................................................... 93 Deletion of the 200-bp promoter domain results in cytokinin-insensitive expression of PIN genes ......................................................................................................................... 93 Uncoupling PIN1 and PIN7 transcription from the cytokinin control modulates plant development and sensitivity to cytokinin .......................................................................... 96 Cytokinin response factors (CRFs) control PIN transcription by direct interaction with the PCRE elements ........................................................................................................... 98 Expression of PIN1 and PIN7 genes is altered in crf mutants ......................................... 99 Loss-of-function mutants of CRFs exhibit an auxin transport-defective phenotype ...... 103 Conclusions .................................................................................................................... 105 Material and methods ...................................................................................................... 106 Plant material and growth condition ............................................................................

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