ISOLATION AND MOLECULAR CHARACTERIZATION OF GENES ASSOCIATED WITH SHOOT REGENERATION OF MUSTARD (BRASSICA JUNCEA) IN VITRO GONG HAIBIAO NATIONAL UNIVERSITY OF SINGAPORE 2003 ISOLATION AND MOLECULAR CHARACTERIZATION OF GENES ASSOCIATED WITH SHOOT REGENERATION OF MUSTARD (BRASSICA JUNCEA) IN VITRO GONG HAIBIAO (M.Sc. SJTU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS I would like to express my utmost gratitude to my supervisor, Assoc. Prof. Pua Eng Chong for his invaluable advice, guidance and support throughout my research work over the past several years. I would also like to extend my sincere thanks to my fellow colleagues in Plant Genetic Engineering Laboratory, Carol, Cheng Wei, Emily, Francis, Huiping, Liu Pei, Mo Hua, Serena, Shuzhen, Wenwei, Yuxia and Dr. Liu Jianzhong for creating a helpful and joyful working environment. I appreciate the help and advice from my friends in other laboratories, Yu Hao, Shuhua, Fuquan and Zhilong. Last but not least, I would like to thank my family, especially my wife, Tong Li, for their love, encouragement and constant support, without which completion of this project would not have been possible. This thesis is also dedicated to my lovely son, Chuwei, who always brings me laughter, joy and strength. i TABLE OF CONTENTS Pages Title Acknowledgements i Table of contents ii List of abbreviations vii List of tables xi List of figures xii Summary xv General introduction Literature review 2.1 Occurrence and regulation of morphogenic events in vitro 2.1.1 Somatic and microspore embryogenesis 2.1.2 Organogenesis 5 2.2 Plant morphogenesis in vitro in relation to ethylene 2.2.1 Regulation of ethylene biosynthesis and action 2.2.1.1 Ethylene biosynthesis 2.2.1.2 Ethylene signal transduction pathway 2.2.1.3 Relationship between ethylene and other signaling pathways 2.2.2 Role of ethylene in plant morphogenesis 2.2.2.1 Shoot organogenesis 2.2.2.2 Somatic embryogenesis (SE) 8 10 12 14 14 16 2.3 Metabolic linkage between ethylene and polyamines (PAs) 2.3.1 PA metabolism 2.3.2 Role of PAs in plant morphogenesis in vitro 2.3.2.1 Shoot organogenesis 2.3.2.2 Somatic embryogenesis 16 17 19 19 20 2.4 Stress in relation to ethylene and PAs 2.4.1 Effects of ethylene 2.4.2 Effects of PAs 2.4.3 Oxidative stress 2.4.3.1 Occurrence and scavenging of ROS 2.4.3.2 H2O2 signaling 2.4.4 Plant regeneration in relation to stress/ H2O2 22 22 23 24 24 28 30 ii 2.5 Genetic control of regeneration 2.5.1 Genotypic variability in Brassica spp. 2.5.2 Genes involved in shoot organogenesis 2.5.3 Genes involved in SE 32 32 33 35 Materials and methods 38 3.1 Plant materials 3.1.1 Brown mustard (Brassica juncea (L.) Czern and Coss) 3.1.2 Arabidopsis thaliana 38 38 38 3.2 Chemical treatments 39 3.3 Shoot regeneration from cultured explants 39 3.4 Gene cloning 3.4.1 Cloning of polymerase chain reaction (PCR)-amplified products 3.4.2 Bacterial transfection 3.4.3 Plasmid DNA isolation 3.4.4 DNA sequencing and analysis 41 41 41 43 44 3.5 Probe labeling 3.5.1 DNA probes 3.5.2 RNA probes 44 44 46 3.6 Genomic DNA isolation and Southern analysis 46 3.7 RNA isolation and northern blot analysis 3.7.1 TRI REAGENT method 3.7.2 CTAB method 3.7.3 Northern blot 47 47 48 48 3.8 mRNA differential display 49 3.9 Quantitative Reverse Transcription PCR (RT-PCR) 50 3.10 Cloning of Full-length cDNA by rapid amplification of cDNA ends (RACE) 3.10.1 5’-RACE 3.10.2 3’-RACE 3.10.3 Generation of full-length cDNA sequences 51 51 52 52 3.11 Cloning of the promoter sequence 3.11.1 Construction of GenomeWalker libraries 3.11.2 Cloning of BjGSTF2 and SRKKK promoters by Genome Walking strategy 53 53 53 3.12 Construction of chimeric genes 3.12.1 Generation of sense and antisense constructs 55 55 iii 3.12.2 Generation of double-stranded construct 3.12.3 Generation of BjGSTF2 promoter:GUS fusions 56 58 3.13 Genetic transformation of Arabidopsis plants 58 3.14 Biochemical analysis 3.14.1 Histochemical assays for the GUS activity 3.14.2 GUS fluorometric assay 3.14.3 Ethylene measurement 3.14.4 Histochemical detection of H2O2 61 61 62 62 63 3.15 Bioinformatics tools used for sequence analysis 63 Identification and expression of genes associated with shoot regeneration from leaf disc explants of mustard (Brassica juncea) in vitro 65 4.1 Introduction 65 4.2 Results 4.2.1 Shoot regeneration from leaf disc explants 4.2.2 Identification of cDNAs differentially expressed in tissues 4.2.3 Categorization of cDNAs 4.2.4 Characterization of cDNAs 4.2.5 Effect of H2O2 4.2.6 Endogenous H2O2 content in tissues during culture 68 68 68 71 76 79 81 4.3 Discussion 84 The cloning of a phi class glutathione S-transferase gene and effects of regulation of its expression on shoot regeneration and stress response 90 5.1 Introduction 90 5.2 Results 5.2.1 Cloning and sequence analysis of GST genes from mustard 5.2.2 Multiple members in the mustard genome and their relationship with other phi class GSTs 5.2.3 Expression of GST genes in different mustard organs 5.2.4 GST expression in response to various treatments 5.2.5 Generation of transgenic plants expressing sense, antisense and double-stranded GST cDNAs 5.2.6 Flowering and stress response in transgenic plants 5.2.7 Shoot regeneration response and ethylene production of cultured tissues in relation to GST expression 93 93 5.3 Discussion 93 103 103 108 110 113 119 iv 5.3.1 Characteristics of GSTs 5.3.2 GST expression and its regulation 5.3.3 Changes in GST expression affect stress tolerance and flowering 5.3.4 Role of GSTs in plant morphogenesis in vitro 124 Cloning and characterization of the promoter of a phi class glutathione S-transferase gene by 5’- deletion analysis 127 6.1 Introduction 127 6.2 Results 6.2.1 Molecular cloning of the BjGSTF2 promoter 6.2.2 Generation of transgenic plants expressing the GUS gene conferred by different BjGSTF2 promoters 6.2.3 Organ specific expression 6.2.4 Changes in the transgene activity conferred by different BjGSTF2 promoters in response to H2O2, ACC, SA and spermidine 6.2.5 H2O2 accumulation and GUS activity in leaf discs during shoot regeneration in vitro 129 129 129 6.3 119 120 122 Discussion 6.3.1 Characterization of BjGSTF2 promoter 6.3.2 Spatial gene expression in transgenic plants 6.3.3 Transgene expression in response to treatments 6.3.4 GUS expression is associated with H2O2 accumulation in transgenic tissues 131 133 134 138 138 138 140 143 Isolation and characterization of a Raf-related MAPKKK gene from mustard 145 7.1 Introduction 145 7.2 Results 7.2.1 Cloning and sequence analysis of SRKKK gene 7.2.2 SRKKK is a homolog of MAPKKK 7.2.3 Expression of SRKKK during shoot regeneration 7.2.4 SRKKK expression in different organs 7.2.5 SRKKK expression in response to treatments 7.2.6 Generation of transgenic plants expressing sense SRKKK 7.2.7 Selection of Arabidopsis AtSRKKK mutants 7.2.8 Expression of PDF1.2, AtVSP and AtGSTF2 in SRKKK overexpressor and mutant 7.2.9 Effects of high concentrations of hormone/chemical treatments on root growth of SRKKK overexpressor and mutant 149 149 153 158 161 161 164 166 168 Discussion 7.3.1 SRKKK encodes a putative Raf-related kinase protein 172 172 7.3 170 v 7.3.2 7.3.3 7.3.4 Spatial and temporal expression of SRKKK Correlation with jasmonate (JA) Correlation with GSH General discussion and conclusion References 173 174 176 179 189 vi LIST OF ABBREVIATIONS 2,4-D 2,4-dichlorophenoxyacetic acid 2-iP 6-(γ,γ-dimethylallylamino)purine ABA abscisic acid ACC 1-aminocyclopropane-1-carboxylate ACO ACC oxidase ACS ACC synthase ADC arginine decarboxylase Agm agmatine Arg arginine AgNO3 silver nitrate APP 1-(aminopropyl)pyrroline APX ascorbate peroxidase A. tumefaciens Agrobacterium tumefaciens AVG aminoethoxyvinylglycine BA benzyladenine bp base pair(s) CaMV cauliflower mosaic virus cDNA complementary deoxyribonucleic acid CAT catalase CM control medium CTAB hexadecyl trimethyl-ammonium bromide DAO diamine oxidase DAB 3,3-diaminobenzidine DAP 1,3-diaminopropane Dc-SAM decarboxylated S-adenosylmethionine DDRT-PCR differential display RT-PCR DEPC diethyl-pyrocarbonate DFM difluoromethylarginine DFMO difluoromethylornithine Dc-SAM decarboxylated SAM DEPC diethy-pyrocarbonate vii DHA dehydroascorbate DHAR DHA reductase DIG digoxigenin DMSO dimethyl sulfoxide DMTU dimethylthiourea DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate E.coli Escherichia coli EDTA ethylene-diamine-tetra-acetate EST expressed sequence tag g gram(s) GA gibberellic acid GABA γ-aminobutyric acid GM germination medium GR glutathione reductase GSH glutathione GSSG glutathione disulphide GST glutathione S-transferase GUS β-glucuronidase h 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Plant Cell 5: 1411-1423 213 [...]... studies, in which downregulation of ACC oxidase by expressing an antisense ACC oxidase RNA in transgenic mustard (Brassica juncea) (Pua and Lee, 1995) and melon (Amor et al., 1998) resulted in a marked reduction in ethylene production and a marked enhancement in shoot morphogenesis in vitro However, the promoting effect of ethylene inhibitors on shoot regeneration of Chinese cabbage (Chi et al., 1994) and. .. expressing a 35S-SRKKK chimeric gene 165 Figure 41 Arabidopsis mutant plants carrying T-DNA insertions within the AtSRKKK gene 167 Figure 42 Expression of PDF1.2, AtVSP and AtGSTF2 genes in SRKKK overexpresser and mutant 169 xiv SUMMARY The main aims of this study were to identify and characterize genes associated with shoot regeneration of mustard in vitro These were achieved by the isolation of 87... the initiation of shoot organogenesis and somatic embryogenesis has yet to be elucidated Although the regulatory role of various factors, notably auxin and cytokinin, on plant morphogenesis in vitro has been well documented, there has been increasing evidence showing that various morphogenic events may be associated with ethylene and polyamines (PAs) (Pua, 1999) Ethylene and PAs are ubiquitous in plants... and Chinese kale (Pua et al., 1996) can be mimicked by exogenous PAs The implication of PAs in shoot regeneration has also been demonstrated in transgenic tobacco plants, where expressing of a human SAMDC cDNA resulted in an increase in the spermidine content and shoot regenerability of the cultured explants (Noh and Minocha, 1994) The findings have prompted the speculation that increased shoot regeneration. .. 2003), and IRE1 (Cary et al., 2001) As part of the long-term goal in this laboratory to elucidate the underlying molecular mechanism that regulates shoot morphogenesis in vitro, the objectives of this study are: (1) To isolate cDNAs associated with shoot morphogenesis of mustard in vitro using mRNA differential display (2) To characterize some cloned genes that express differentially in poorly and highly... tissues (3) To investigate the role of some cloned genes in shoot regeneration by overexpression and downregulation of these genes in transgenic plants 3 (4) To investigate the molecular mechanism as to how morphogenesisrelated genes are regulated by cloning and functional analysis of the gene promoter 4 2 LITERATURE REVIEW 2.1 Occurrence and regulation of morphogenic events in vitro Since the first... ethylene and PAs play an important role in shoot morphogenesis of a wide range of plant species in vitro (Pua 1999) Shoot regeneration of cultured explants can be greatly enhanced by inhibition of ethylene production or action using aminoethoxyvinylglycine (AVG) and AgNO3, respectively (Chi et al., 1991; Palmer, 1992; Pua and Chi, 1993) The regulatory role of ethylene has also been supported by results of. .. two-component histidine kinases involved in sensing environmental stimuli Currently, five members of this family in Arabidopsis, including ETR1, ERS1, ETR2, EIN4 and ERS2, have been identified (Chang et al., 1993; Hua et al., 1995; Hua and Meyerowitz, 1998; Sakai et al., 1998) ETR1 possesses a modular structure containing an ethylene-binding domain in the Nterminus (Schaller and Bleecker, 1995) and regions... the induced expression of PDF1.2 because pretreatment of plants with ascorbic acid blocked the induction (Surplus et al., 1998) These results, together with the findings that induction of PDF1.2 was also inhibited in mutants defective in ethylene and JA signaling (Wang et al., 2002), suggest that ROS lie upstream of the ethylene and JA pathways 2.2.2 Role of ethylene in plant morphogenesis 2.2.2.1 Shoot. .. rapidly induced by ethylene Overexpression of ERF1 in an ein3 background leads to constitutive activation of some ethylene responses (Solano et al., 1998) Interestingly, the EIN3 homodimers can bind to a specific target sequence in the promoter of ERF1 gene, resulting in upregulation of ERF1 expression These findings provide a functional link of ERF1 to the downstream of the ethylene signaling pathway . NATIONAL UNIVERSITY OF SINGAPORE 2003 ISOLATION AND MOLECULAR CHARACTERIZATION OF GENES ASSOCIATED WITH SHOOT REGENERATION OF MUSTARD (BRASSICA JUNCEA) IN VITRO . ISOLATION AND MOLECULAR CHARACTERIZATION OF GENES ASSOCIATED WITH SHOOT REGENERATION OF MUSTARD ( BRASSICA JUNCEA ) IN VITRO GONG HAIBIAO. expression and its regulation 120 5.3.3 Changes in GST expression affect stress tolerance and 122 flowering 5.3.4 Role of GSTs in plant morphogenesis in vitro 124 6 Cloning and characterization of