CLONING AND CHARACTERIZATION OF DIAMINE OXIDASE AND GLUTAMATE DECARBOXYLASE GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR ROLES IN SHOOT MORPHOGENESIS IN VITRO JIAO YUXIA NATIONAL UNIVERSITY OF SINGAPORE 2004 CLONING AND CHARACTERIZATION OF DIAMINE OXIDASE AND GLUTAMATE DECARBOXYLASE GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR ROLES IN SHOOT MORPHOGENESIS IN VITRO JIAO YUXIA (M.Eng. ) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements I would like to express my deepest gratitude to my supervisor, Associate Professor Pua Eng Chong for his invaluable advice, guidance, inspiration and patience, help and support over the past years. I would also like to extend my sincere thanks to all my friends in the laboratory, Carol Han Ping, Francis Tan Chee Kuan, Serena Lim Tze Soo, Emily Tay Boon Hui, Gong Haibiao, Hu wenwei, Cheng Wei, Mo Hua, Xu Yifeng, Yang Shuhua, Wang Yu, Teo Lai Lai and Dr. Yu Hao, for their help and encouragement during my staying in Singapore. Their presence has created an enjoyable and productive working environment and made my staying in Singapore an unforgettable memory. Finally, I would like to thank my family members. My parents and siblings have been an everlasting source of power throughout my life. Without their support and encouragement, I could not go so far in my education and academic career. At last but not the least, I should owe my thanks to my husband Liu Feng, to whom this thesis is dedicated. Besides love, care, encouragement and patience, as a fellow, he also gives me professional support, help and inspiration. i Table of Contents Page Acknowledgements i Table of Contents ii List of Abbreviations x List of Figures xiii List of Tables xvii Summary xviii Introduction Literature Review Metabolism and regulation of PAs 2.1.1 PA biosynthesis 2.1 2.1.1.1 ODC 2.1.1.2 ADC 10 2.1.1.3 AIH and CPA 11 2.1.1.4 SAMDC 12 2.1.1.5 SPDS 14 2.1.1.6 SPMS 16 ii 2.1.2 PA catabolism 18 2.1.2.1 DAO 18 2.1.2.1.1 Molecular features and catalytic mechanism 19 2.1.2.1.2 Expression during plant growth and development 21 2.1.2.1.3 Expression in response to external stimuli 22 2.1.2.2 PAO 2.1.3 2.2 Modulation of PAs in transgenic plants 25 28 Roles of PAs in plant morphogenesis in vitro 34 2.2.1 Somatic embryogenesis 34 2.2.2 Shoot morphogenesis 35 2.2.3 Rhizogenesis 38 2.3 Effects of PAs on oxidative stress 39 2.4 GABA shunt pathway 41 2.4.1 GAD 43 2.5 2.4.1.1 Sequence characteristics of plant GADs 43 2.4.1.2 GAD expression 44 2.4.1.3 Regulation of plant GADs 46 2.4.2 GABA-T 48 2.4.3 SSADH 48 Proposed roles of GABA 50 iii 2.5.1 Stress response 50 2.5.2 Plant defense to herbivory 52 2.5.3 Plant growth and development 53 Interaction between PAs, ethylene and GABA 56 Materials and Methods 59 Plant materials 59 3.1.1 Mustard 59 3.1.2 Arabidopsis 59 3.2 Chemical treatments 60 3.3 Shoot regeneration from cultured explants 60 3.4 Gene cloning 62 3.4.1 Cloning of PCR-amplified products 62 3.4.2 Bacterial transfection 62 3.4 Plasmid DNA isolation 63 3.4.4 DNA sequencing and analysis 64 Probe labeling 65 3.5.1 DNA probes 65 3.5.2 RNA probes 66 Isolation of cDNA clones 66 2.6 3.1 3.5 3.6 iv 3.6.1 Library titering 66 3.6.2 Library screening 67 3.6.3 In vivo excision 68 3.7 Genomic DNA isolation and Southern analysis 68 3.8 RNA isolation and northern blot analysis 70 3.9 cDNA synthesis by reverse transcription 71 3.10 Quantitative reverse transcription PCR (RT-PCR) 71 3.11 Cloning of full-length cDNA by RACE 72 3.11.1 5’-RACE 72 3.11.2 3’-RACE 74 3.11.3 Generation of full-length cDNA sequences 74 Cloning of the BjDAO promoter 74 3.12.1 Construction of libraries 74 3.12.2 Promoter cloning by Genome Walking strategy 75 Construction of chimeric genes 76 3.13.1 DAO-GFP fusion protein 76 3.13.2 Sense and antisense DAO 78 3.13.3 Sense, antisense and dominant-negative GAD 78 3.13.4 Generation of BjDAO promoter::GUS fusions 81 Genetic transformation of plants 84 3.12 3.13 3.14 v 3.14.1 Mustard 84 3.14.2 Arabidopsis 85 Biochemical analysis 87 3.15.1 GFP detection with confocal microscopy 87 3.15.2 Histochemical assays for the GUS activity 87 3.15.3 GUS fluorometric assay 87 3.15.4 Ethylene measurement 88 3.15.5 Assays for endogenous PAs 88 3.15.6 Histochemical detection of H2O2 89 Bioinformatics tools used for sequence analysis 90 Cloning and characterization of DAO gene and promoter 91 4.1 Introduction 91 4.2 Results 94 4.2.1 Molecular cloning of DAO gene and promoter 94 4.2.2 Sequence analysis of DAO gene 97 4.2.2 Subcellular localization of BjDAO 107 4.2.4 Genomic Southern analysis 111 4.2.5 DAO expression during germination 113 4.2.6 Sequence analysis of DAO promoter 113 3.15 3.16 vi 4.2.7 Functional analysis of DAO promoter 120 4.2.8 Gene expression conferred by different promoters in response to external stimuli 125 Discussion 128 4.3.1 Characteristics of DAO 128 4.3.2 Molecular characterization of the 5’-upstream regulatory sequence of DAO 132 4.3.3 Light-regulated DAO expression 137 4.3.6 Regulation of DAO expression in response to stress 138 Cloning and characterization of GAD genes 142 5.1 Introduction 142 5.2 Results 145 5.2.1 Cloning of GAD genes 145 5.2.2 Sequence analysis of GAD 147 5.2.3 Genomic Southern analysis 159 5.2.4 Spatial and temporal GAD expression 159 5.2.5 GAD expression in response to external stimuli 163 5.2.5.1 Effects of phytohormones 163 5.2.5.2 Effects of paraquat and H2O2 166 5.2.5.3 Effects of NaCl and mannitol 166 4.3 vii 5.2.5.4 Effects of exogenous glutamate and GABA 171 5.2.5.5 Effects of CaCl2 and pH 171 5.2.5.6 Effects of temperature 177 5.2.6 GAD member specific expression in different organs 177 5.2.7 Differential expression of GAD in response to external stimuli 182 Discussion 182 5.3.1 Characteristics of GAD genes 184 5.3.2 Spatial and temporal expression of GAD 186 5.3.3 GAD expression in response to exogenous stimuli 188 Effects of overexpression and downregulation of DAO and GAD RNAs on shoot regeneration in vitro 192 6.1 Introduction 192 6.2 Results 194 6.2.1 Construction of sense and antisense DAO and GAD genes 194 6.2.2 Production of transgenic plants 194 6.2.3 Characterization of transgenic plants 200 6.2.4 Ethylene production in transgenic plants 203 6.2.5 Cellular PA content in transgenic plants 206 6.2.6 H2O2 production in transgenic plants 209 6.2.7 Shoot regeneration response of transgenic plants 212 5.3 viii Langebartels C, Kerner KJ, Leonardi S, Schraudner M, Trost M, Heller W, 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Annu Rev Plant Physiol Plant Mol Biol 49: 697-725 267 [...]... involved in shoot regeneration in vitro This was achieved by the cloning and characterization of diamine oxidase (DAO) and glutamine decarboxylase (GAD) genes from mustard The genomic (gBjDAO) and cDNA (BjDAO) clones of DAO were isolated from mustard gBjDAO consisted of 5’ upstream regulatory sequences and 4 exons interrupted by 3 introns, and the protein-coding sequence was identical to the open reading... accumulating evidence showing that polyamines (PAs), including putrescine (Put), spermidine (Spd) and spermine (Spm), and its oxidative product, H2O2, are implicated in shoot morphogenesis in vitro and/ or somatic embryogenesis in several plant species However, the mechanism of PA action is not clear The main aim of this study is to investigate whether PA oxidation and its downstream catabolic pathway are involved... binding of PAs to 14-3-3 proteins resulted in a conformational change and facilitated the binding and inhibition of nitrate reductase by 14-3-3 (Athwal and Huber, 2002) PAs have been shown to play an important role in a wide range of physiological processes during plant growth and development, including dormancy breaking of tubers 4 and seed germination (Bagni, 1989), stimulation and development of. .. living organisms (Martin-Tanguy, 2001) The most common aliphatic PAs are the diamine putrescine (Put), triamine spermidine (Spd) and tetraamine spermine (Spm) Put and Spd are generally more abundant, while Spm is present in lower or trace amounts (Bagni and Tassoni, 2001) In addition, diamine cadaverine (Cad) is mainly found in members of the family Fabaceae (Federico and Angelini, 1988) PAs are positively... development in vivo (Evans and Malmberg, 1989) There has been increasing evidence showing that PAs are also implicated in shoot organogenesis in vitro and somatic embryogenesis in several plant species (Pua, 1999) The presence of higher levels of cellular PAs has been associated with increased shoot regeneration from maize callus (Guregue et al., 1997), somatic embryogenesis of eggplant (Sharma and Rajam,... 1999) To investigate whether shoot morphogenesis in vitro is associated with PA catabolism, the objectives of this study are: 1 To clone and characterize the DAO gene from mustard, including the promoter; 2 To clone and characterize the GAD genes from mustard; 3 To manipulate the cellular content of PAs by overexpression and downregulation of the DAO gene in transgenic plants and its effects on shoot. .. Rajam, 1995; Yadav and Rajam, 1998), carrot (Noh and Minocha, 1994) and rice (Bajaj and Rajam, 1995) and rhizogenesis of tobacco (Altamura, 1994) and poplar (Hausman et al., 1997a, 1997b) In addition to PAs, results from several lines of study have shown that shoot regeneration and somatic embryogenesis can be enhanced by inhibition of synthesis or action of ethylene (Pua, 1999; Pua and Gong, 2004),... 2-aminoethoxyvinyl glycine BA Benzyladenine BCIP 5-bromo-4-chloro-2-indolyl-phosphate CHA cyclohexylamine DAB 3,3-diaminobenzidine Dap diaminopropane dcSAM decarboxylated S-adenosyl methionine DEPC diethyl-pyrocarbonate DFMA difluoromethylarginine DFMO difluoromethylornithine DIG digoxigenin DMSO dimethyl sulfoxide FAD flavin adenine dinucleotide GA3 gibberellins acid GABA γ-aminobutyric acid IBA indole-3-butyric... Schematic diagram of GFP and DAO fusion constructs in pGreenGFP 77 4 Construction of DAO-AS and DAO-S 79 5 Construction of chimeric genes consisting of GADS, GAD-AS and tGAD 80 6 Schematic representation of constructs carrying the gene fusions of the GUS coding sequence under 35S promoter with DAO 5’UTR behind 82 7 Schematic representation of constructs carrying the gene fusions of the GUS coding sequence... deduced amino acid sequences of mGAD2 149 29 Comparison of the deduced amino acid sequence between mGADs and other plant GAD homologs 152 30 Comparison of the deduced amino acid sequence between mGAD2 and GADs from non-plant species 154 31 Alignment of nucleotide sequence of the 5’- and 3’-termini of mustard GADs 156 32 Prediction of the secondary structure of C-terminal of mGADs with PSIPRED software . UNIVERSITY OF SINGAPORE 2004 CLONING AND CHARACTERIZATION OF DIAMINE OXIDASE AND GLUTAMATE DECARBOXYLASE GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR ROLES IN SHOOT MORPHOGENESIS IN VITRO. CLONING AND CHARACTERIZATION OF DIAMINE OXIDASE AND GLUTAMATE DECARBOXYLASE GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR ROLES IN SHOOT MORPHOGENESIS IN VITRO . pathway are involved in shoot regeneration in vitro. This was achieved by the cloning and characterization of diamine oxidase (DAO) and glutamine decarboxylase (GAD) genes from mustard. The