Regulation of phenotypic plasticity in high ambient temperature: ELF3 and BZR1 as major thermostats gating PIF4 signaling Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät III Agrar-und Ernährungswissenschaften, Geowissenschaften und Informatik der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Carla Ibáñez Robles Geb. am 24.11.1988 in Valencia (Spanien) Halle, 2017 A mis padres Abstract – English Version Together with light, temperature is one of the major environmental cues regulating plant growth. In Arabidopsis thaliana, growth responses to high ambient temperature occur already in early stages of seedling development, hypocotyl elongation being one of the best characterized model phenotypes. At the molecular level, temperature-mediated plant morphology (also named thermomorphogenesis) is largely dependent on the transcription factor PHYTOCHROME INTERACTOR FACTOR 4 (PIF4). However, regulation of PIF4 on both transcriptional and post- transcriptional level is very complex and remains rather poorly understood. To exploit both the phenotypic and genotypic variation observed and to identify novel components of the temperature signaling pathway, temperature-induced hypocotyl elongation (TIHE) was used as a model response to perform a QTL analysis in the Bay x Sha RIL population and an EMS-mutagenesis screen in the Rrs-7 ecotype. As a result, the circadian clock component EARLY FLOWERING 3 (ELF3) and the brassinosteroid transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) are highlighted here as novel components by gating PIF4 in the control of thermomorphogenesis. Key words: Arabidopsis, ecotype, hypocotyl, thermomorphogenesis, PIF4, brassinosteroids, BZR1, auxin, ELF3, temperature, signaling. Abstract – Deutsche Version Neben Licht ist Temperatur einer der wichtigsten Umwelteinflüsse, die pflanzliches Wachstum regulieren. Das Wachstum von Arabidopsis thaliana reagiert bereits in den frühen Stadien der Keimlingsentwicklung sehr sensitiv auf erhöhte Umgebungstemperaturen. Hypokotylelongation ist hierbei einer der am besten charakterisierten Phänotypen. Auf molekularer Ebene hängt die temperaturregulierte Morphologie (Thermomorphogenese) zu großen Teilen vom Transkriptionsfaktor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) ab. Die Regulation von PIF4 ist jedoch sowohl auf transkriptioneller als auch auf translationeller Ebene sehr komplex und bislang wenig verstanden. Zur Identifizierung neuer Komponenten eines Temperatursignalweges wurde die genotypische und phänotypische Variation temperaturinduzierter Hypokotylelongation als Modelphänotyp in einer QTL-Analyse (Bay x Sha RIL Population) und einem EMS- Mutagenesescreen (Rrs-7 Ökotyp) genutzt. Als Ergebnis dieser Ansätze konnten sowohl eine Komponente der circadianen Uhr, EARLY FLOWERING 3 (ELF3), als auch ein in der Brassinosteroidsignalkette relevanter Transkriptionsfaktor, BRASSINAZOLE RESISTANT 1 (BZR1), identifiziert und ihre Rollen in der temperaturinduzierten Signaltransduktion charakterisiert werden. Stichworte: Arabidopsis, Ökotyp, Hypokotyl, Thermomorphogenese, PIF4, Brassinosteroide, BZR1, Auxin, ELF3, Temperatursignalweges. Namen der Gutachter: Prof. Dr. Marcel Quint and Prof. Dr. Ute Höcker Verteidigungsdatum: 10.07.2017 Index 1. Introduction .............................................................................................................. 1 1.1 Effect of globally increasing ambient temperature on plant performance .......................... 1 1.2 Thermomorphogenesis .................................................................................................... 1 1.3 Temperature sensing in plants ......................................................................................... 2 1.4 The temperature signal transduction ................................................................................ 4 1.5 The relevance of brassinosteroids in growth-associated responses ................................ 6 1.5.1 The brassinosteroid signaling network ...................................................................... 6 2. Objectives ................................................................................................................ 8 3. Materials and Methods ........................................................................................... 9 3.1 Escherichia coli ................................................................................................................ 9 3.1.1 E.coli strains ............................................................................................................. 9 3.1.2 Luria-Bertani (LB) medium ....................................................................................... 9 3.1.3 Growth conditions ..................................................................................................... 9 3.1.4 Heat shock transformation and selection of positive clones ...................................... 9 3.2 Agrobacterium tumefaciens............................................................................................ 10 3.2.1 A.tumefaciens strain ............................................................................................... 10 3.2.2 Growth media ......................................................................................................... 10 3.2.3 Growth conditions ................................................................................................... 10 3.2.4 Heat shock transformation and selection ................................................................ 10 3.3 Arabidopsis thaliana ....................................................................................................... 10 3.3.1 Seeds sterilization and stratification ........................................................................ 10 3.3.2 Growth conditions ................................................................................................... 10 3.3.3 Hormone/Inhibitor treatments ................................................................................. 11 3.3.4 Temperature-Induced Hypocotyl Elongation (TIHE) assay ...................................... 11 3.3.5 Chlorophyll quantification ........................................................................................ 11 3.3.6 Flowering time estimation ....................................................................................... 12 3.3.7 Rosette and petiole measurements ........................................................................ 12 3.3.8 Arabidopsis thaliana transformation by floral dip ..................................................... 12 3.3.9 Selection of transformed Arabidopsis thaliana seedlings ........................................ 12 3.4 Molecular biology methods ............................................................................................ 13 3.4.1 Semi-quantitative PCR ........................................................................................... 13 3.4.2 Genomic DNA extraction for mapping ..................................................................... 13 3.4.2.1 Extraction buffer .............................................................................................. 13 I 3.4.2.2 Protocol........................................................................................................... 14 3.4.3 Mapping by CAPS markers .................................................................................... 14 3.4.4 RNA Extraction, cDNA Synthesis, and qRT- PCR ................................................... 14 3.4.5 Protoplast assay by the Tape-Arabidopsis Sandwich method ................................. 15 3.4.5.1 Solutions ......................................................................................................... 15 3.4.5.2 Protoplast preparation ..................................................................................... 15 3.4.5.3 Protoplast transformation ................................................................................ 16 3.4.5.4 Luciferase assay ............................................................................................. 16 3.4.5.5 GUS assay ...................................................................................................... 16 3.4.5.6 LUC normalization .......................................................................................... 17 3.4.6 Western blot ........................................................................................................... 17 3.4.6.1 Protein extraction ............................................................................................ 17 3.4.6.2 SDS-PAGE electrophoresis ............................................................................. 18 3.4.6.3 Electrotransfer (semi-dry transference) ........................................................... 18 3.4.7 Chromatin-immunoprecipitation-PCR (ChIP-PCR) .................................................. 18 3.4.7.1 Buffers/Material ............................................................................................... 18 3.4.7.2 Protocol........................................................................................................... 19 3.4.8 Golden Gate cloning ............................................................................................... 22 3.5 Statistics ........................................................................................................................ 22 3.5.1