Characterisation of selected Arabidopsis aldehyde dehydrogenase genes: role in plant stress physiology and regulation of gene expression Dissertation Zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Tagnon Dègbédji MISSIHOUN aus Cotonou, Benin Bonn, November 2010 Angefertigt mit Genehmigung der Mathematisch- Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes 1. Referentin: Prof. Dr. Dorothea Bartels 2. Koreferent: Priv. Doz. Dr. Hans-Hubert Kirch Tag der Promotion: 22. Februar 2011 Erscheinungsjahr: 2011 II DECLARATION I hereby declare that the whole PhD thesis is my own work, except where explicitly stated otherwise in the text or in the bibliography. Bonn, November 2010 ------------------------------------ Tagnon D. MISSIHOUN III DEDICATION To My wife: Fabienne TOSSOU-MISSIHOUN and our kids Floriane S. Jennifer and Sègnon Anges- Anis My parents: Lucrèce KOTOMALE and Dadjo MISSIHOUN My sister and brothers: Mariette, Marius, Ricardo, Renaud, Ulrich And my dearest aunts and uncles: Hoho, Rebecca, Cyriaque, Dominique, Alphonsine IV CONTENTS ABBREVIATIONS ...............................................................................................................................................X FIGURES AND TABLES ...............................................................................................................................XIII SUMMARY ........................................................................................................................................................... 1 1. INTRODUCTION ....................................................................................................................................... 3 1.1 Climate changes and environmental stress...................................................................................... 3 1.2 Plant stress and mechanisms of tolerance........................................................................................ 3 1.3 Gene products related to abiotic stress ............................................................................................ 4 1.3.1 Regulatory pathways of stress-related gene expression in plants............................................... 5 1.3.1.1 Osmotic/oxidative stress signalling ....................................................................................... 5 1.3.1.2 Ca2+-dependent signalling...................................................................................................... 6 1.3.2 ABA signalling........................................................................................................................... 7 1.3.2.1 ABA metabolism ................................................................................................................... 7 1.3.2.2 ABA perception..................................................................................................................... 7 1.3.2.3 ABA signal transduction........................................................................................................ 8 1.3.3 Stress inducible proteins and other compounds........................................................................ 10 1.3.3.1 LEA proteins....................................................................................................................... 10 1.3.3.2 Compatible solutes............................................................................................................... 11 1.3.3.2.1 Mannitol, D-ononitol and sorbitol ................................................................................ 11 1.3.3.2.2 Trehalose ...................................................................................................................... 12 1.3.3.2.3 Sucrose ......................................................................................................................... 12 1.3.3.2.4 Fructans ........................................................................................................................ 13 1.3.3.2.5 Proline .......................................................................................................................... 13 1.3.3.2.6 Glycine betaine............................................................................................................. 14 1.3.3.3 Small RNAs......................................................................................................................... 15 1.3.3.4 Reactive Oxygen Species (ROS) ......................................................................................... 15 1.3.3.5 Aldehydes and the peroxidation of membrane lipids........................................................... 16 1.3.3.6 Aldehyde dehydrogenases (ALDHs) as ROS-detoxifying enzymes.................................... 17 1.3.3.7 Aldehyde dehydrogenase genes........................................................................................... 18 1.3.3.8 Betaine aldehyde dehydrogenases ....................................................................................... 19 1.3.3.9 Aminoaldehyde dehydrogenases and the polyamine metabolism........................................ 20 1.4 Objectives of the study..................................................................................................................... 20 2. MATERIALS AND METHODS.............................................................................................................. 22 2.1 Materials........................................................................................................................................... 22 2.1.1 Plant materials .......................................................................................................................... 22 2.1.2 Chemicals ................................................................................................................................. 22 2.1.3 DNAs, vectors and bacteria...................................................................................................... 22 2.1.3.1 cDNAs ................................................................................................................................. 22 2.1.3.2 Vectors................................................................................................................................. 23 V 2.1.3.2.1 pJET1.2......................................................................................................................... 23 2.1.3.2.2 pBT10-GUS.................................................................................................................. 23 2.1.3.2.3 pRTL2-GUS vector ...................................................................................................... 23 2.1.3.2.4 pGJ280.......................................................................................................................... 23 2.1.3.2.5 pET28a ......................................................................................................................... 23 2.1.3.2.6 pBIN19 and pROK2 ..................................................................................................... 24 2.1.3.2.7 pPG-Tkan ..................................................................................................................... 24 2.1.3.3 Bacteria................................................................................................................................ 24 2.1.4 Enzymes and DNA-marker ...................................................................................................... 24 2.1.5 Software, programs and online tools ........................................................................................ 25 2.1.6 Machines and other devices...................................................................................................... 25 2.1.7 Membranes ............................................................................................................................... 26 2.1.8 Kits ........................................................................................................................................... 26 2.1.9 Media, buffers and solutions .................................................................................................... 26 2.1.9.1 Media................................................................................................................................... 26 2.1.9.2 Buffers and solutions ........................................................................................................... 27 2.2 Methods............................................................................................................................................. 28 2.2.1 Growth conditions .................................................................................................................... 28 2.2.1.1 Seed culture and plant growth ............................................................................................. 28 2.2.1.2 Growth of microorganisms .................................................................................................. 29 2.2.2 Primers ..................................................................................................................................... 29 2.2.3 Extraction of nucleic acids ......................................................................................................