Technische Universität München Lehrstuhl für Botanik Regulation of the abscisic acid response by protein phosphatase 2C-interacting proteins ABP7 and ABP9 in Arabidopsis thaliana Yue Ma-Lauer Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. R. Hückelhoven Prüfer der Dissertation: 1. Univ.-Prof. Dr. E. Grill 2. apl. Prof. Dr. R. A. Torres Ruiz Die Dissertation wurde am 07.12.2010 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 21.03.2011 angenommen. Content Content Abbreviations................................................................................................ 1 Summary ....................................................................................................... 6 Zusammenfassung ....................................................................................... 9 1. Introduction............................................................................................. 12 1.1 ABA signal ........................................................................................... 12 1.2 Pathogenesis-Related Proteins ......................................................... 24 1.3 The family of ABP7 and ABP9 in Arabidopsis thaliana ......................... 30 1.4 The aim of this work............................................................................. 31 2. Materials and methods ........................................................................... 34 2.1 Materials .............................................................................................. 34 2.1.1 Vectors and primers (refer to Appendix) ......................................... 34 2.1.2 Plant materials ............................................................................... 34 2.1.3 Micro-organisms ............................................................................ 34 2.1.4 Reagents ....................................................................................... 35 2.1.5 Media ............................................................................................ 35 2.1.6 Buffers ........................................................................................... 39 2.1.7 Antibodies ...................................................................................... 40 2.2 Methods............................................................................................... 40 2.2.1 Standard molecular biology methods ............................................. 40 2.2.1.1 DNA analysis ........................................................................... 40 2.2.1.2 RNA analysis ........................................................................... 41 2.2.2 Protein analysis ............................................................................. 42 2.2.2.1 Expression and purification of proteins .................................... 42 I Content 2.2.2.2 Protein extraction from Arabidopsis thaliana ............................ 44 2.2.2.3 Determination of protein concentrations by Bradford assay ..... 45 2.2.2.4 Protein detection by Western blot ............................................ 46 2.2.2.5 -Glucuronidase activity assay ................................................ 48 2.2.2.6 Protein phosphatase 2C enzyme activity assay ....................... 48 2.2.2.7 Assays to identify binding ligand(s) of ABP7 and ABP9 ........... 49 2.2.2.7.1 Refolding of the denatured protein ..................................... 49 2.2.2.7.2 Binding test between ABP9 and phytohormones ............... 51 2.2.3 Transient gene expression in protoplasts ....................................... 51 2.2.3.1 Protoplasts preparation ........................................................... 51 2.2.3.1.1 Isolation of protoplasts from leaves of Arabidopsis thaliana51 2.2.3.1.2 Protoplasts preparation from Arabidopsis cell cultures ....... 53 2.2.3.2 Protoplast transfection ............................................................. 54 2.2.3.3 Activity assay of the reporter genes ......................................... 55 2.2.3.4 Signal detection by fluorescence microscopy and confocal microscopy .......................................................................................... 56 2.2.4 Analysis of plants ........................................................................... 56 2.2.4.1 Transformation of Arabidopsis ................................................. 56 2.2.4.2 Physiological assays of transgenic plants ................................ 57 2.2.4.3 Histochemical staining of transgenic Arabidopsis .................... 58 2.2.5 Computer analysis of DNA sequences, data, and pictures ............. 59 3. Results .................................................................................................... 60 3.1 Functional characterization of ABP7/9’s role in ABA signal transduction in the transient expression system ................................................................ 60 3.1.1 ABP7 and ABP9 overexpressors and ABA signal transfer .............. 60 II Content 3.1.2 Effect of ABP7 and ABP9 overexpression on ABA responses in dependence of ABI1 and ABI2 ................................................................ 69 3.1.3 Knockdown of ABP7 and ABP9 expression by RNA interference and ABA responses ....................................................................................... 71 3.2 Regulation of phytohormone responses in transgenic Arabidopsis by ABP7 and ABP9 ........................................................................................ 73 3.2.1 Physiological analysis of ABA responses in Arabidopsis with deregulated expression of ABP7 and ABP9 ............................................ 73 3.2.2 Phenotypes of transgenic lines in respect to exogenous phytohormones....................................................................................... 80 3.2.2.1 ABA ......................................................................................... 80 3.2.2.2 Auxin ....................................................................................... 82 3.2.2.3 Cytokinin and ethylene ............................................................ 87 3.3 Expression studies of ABP9 ................................................................. 91 3.3.1 Histochemical staining of endogenous promoter::genomic ABP9:: β-glucuronidase fusion transgenic plants ................................................ 91 3.3.2 Regulation of ABP9’s expression ................................................... 92 3.3.2.1 RT-PCR to test the possible regulations by ABA and auxin ...... 92 3.3.2.2 ABP9-GUS expression in response to Agrobacteria ................ 93 3.4 Subcellular localization of ABP7 and ABP9 in Arabidopsis protoplasts. 95 3.5 Physical interaction between ABP7/9 and ABI1/abi1/ABI2/abi2 ........... 96 3.6 Attempts to identify ligand(s) of ABP9 ................................................ 100 3.6.1 Expression and purification of histag proteins in E.coli ................ 100 3.6.2 Analysis of anti-ABP7 and anti-ABP9 antibodies ......................... 103 3.6.3 Binding test between ABP9 and phytohormones ......................... 105 III Content 3.6.3.1 ABP9 and 14C-ABA ................................................................ 106 3.6.3.2 ABP9 and 14C-zeatin.............................................................. 107 3.6.4 Protein phosphatase 2C activity tests of ABI1 and ABI2 in the presence of ABP7 and ABP9 ................................................................... 109 4. Discussion .............................................................................................111 4.1 Roles of ABP7 and ABP9 in ABA signal pathway and in phytohormones crosstalk .................................................................................................. 113 4.2 The ABP7/ABP9’s family and their signal pathways ........................... 121 5. Appendix ............................................................................................... 129 6. References ............................................................................................ 141 Acknowledgments .................................................................................... 169 Curriculum Vitae ....................................................................................... 170 IV Abbreviations Abbreviations ABA abscisic acid ABI ABA insensitive ABRE ABA response element ACC 1-aminocyclopropane-1-carboxylic acid Amp ampicillin AtHB homeodomain protein in Arabidopsis thaliana ATP adenosine 5’-triphosphate BAP 6-benzylaminopurine ß-ME ß-mercaptoethanol ß-gal ß-galactosidase b-HLH-ZIP basic helix-loop-helix leucine zipper BiFC bimolecular fluorescence complementation bp base pair BSA bovine serum albumin bZIP basic-leucine zipper CaMV Cauliflower mosaic virus CE coupling element cDNA complementary deoxyribonucleic acid CHLH H subunit of the magnesium-protoporphyrin-IX chelatase CTAB cetyltrimethylammonium bromide 2,4-D 2,4-dichlorophenoxyacetic acid DEPC diethyl pyrocarbonate DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxyribonucleic triphosphate DRE drought response element 1 Abbreviations DTT dithiotreitol EDTA ethylenediamine tetraacetic acid EGTA ethylene glycol- bis(2-aminoethylether)-N,N,N′,N′-tetraacetic