애기장대 지질 대사에서 Marneral Synthase와 Dark Response Factor1의 기능 분석

다국어 초록 (Multilingual Abstract)

The lipids found in plants are present in various molecular forms such as fatty acid, wax, cutin, terpenoid, sterol and flavonoid. Lipids play roles in several processes such as energy storage, signaling, and defense mechanisms, and are structural components of cell membranes. These molecules are known to play an important role in the normal growth and serve to induce tolerance against diverse biological and non-biological stresses. Among them, terpenoid are important for life and survival and convert structurally diverse triterpenoids in plants. Most triterpenoids use a common biosynthetic intermediate, 2, 3-oxidosqualene (OS), which is cyclized by 2, 3-oxidosqualene cyclase (OSC). In order to understand the function of OSC in Arabidopsis thaliana, firstly, I investigated the MRN1 gene that encodes marneral synthase and is involved in an unusual triterpenoid pathway. I characterized an Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in 35Spro:MRN1 transgenic plants, but not in the wild-type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The GFP:MRN1 protein was localized to the endoplasmic reticulum in tobacco protoplasts. This result provides evidence indicating that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis. Secondly, environmental stress regulates several lipid metabolisms such as accumulation of cuticular waxes, composition alteration of cutin layer, and formation of flavonoid in plants. Yet, how cuticular wax biosynthesis is down-regulated in response to environmental stress is unknown. In order to understand the down-regulation of wax biosynthesis in Arabidopsis, I investigated the DARF gene, which is one of the AP2/ERF transcription factors involved in growth, development, wax formation, and various stress response in plants. I showed that overexpression of the DARF (Dark Response Factor) gene encoding an AP2/ERF-type transcription factor, which is epidermis-preferentially expressed and dark-induced, resulted in alterations in the ultrastructure of cuticular layers and a ~45% reduction of cuticular wax load in Arabidopsis. Bifunctional DARF transcription factor represses the expression of FAR6, CER1, and CHS genes involved in cuticular wax or flavonol/anthocyanin biosynthesis and activates expression of dark-inducible DIN11 and defense-related PDF1.2 genes by directly binding to the consensus GCC-box motifs in their promoters. DARF overexpression lines were more sensitive to water loss and UV-B photo damage compared with wild-type, but more resistant to dark-induced leaf senescence and Alternaria brassicicola infection. In contrast, darf mutants showed opposite phenotypes of DARF overexpression lines under the same stress conditions. This result suggests that metabolic changes for plant adaptation to extended darkness are controlled at the levels of transcription via bifunctional transcription factor. Taken together, this study elucidates the function of lipid metabolism for normal growth and development by cell expansion or elongation and revelas the regulation of lipid metabolism for adaptation to dark response in Arabidopsis.

목차 (Table of Contents)

• ABSTRACT (in English) 1
• CHAPTER 1 General Introduction 4
• 1.1 CONTENTS 5
• 1.1.1 Fatty Acid Metabolism 5
• 1.1.2 Cuticular Wax Metabolism 10
• 1.1.3 Cutin Metabolism 12
• 1.1.4 Terpenoid Metabolism 16
• 1.1.5 Sterol Metabolism 17
• 1.1.6 Flavonoid Metabolism 21
• 1.1.7 The Purposes and Content of This Study 22
• 1.2 REFERENCES 26
• CHAPTER 2 Identification of Marneral Synthase, Which Is Critical for Growth and Development in Arabidopsis 42
• 2.1 ABSTRACT 43
• 2.2 INTRODUCTION 44
• 2.3 MATERIALS AND METHODS 47
• 2.3.1 Plant Materials, Growth Conditions, and Mutant Screening 47
• 2.3.2 Construction of MRN1pro:GUS Transgenic Plant 47
• 2.3.3 Construction of MRN1pro:MRN1gDNA Transgenic Plant 48
• 2.3.4 Analysis by RT-PCR and Quantitative RT-PCR 48
• 2.3.5 Light and Scanning Electron Microscopy 48
• 2.3.6 Semi-thin Sections of SAM 49
• 2.3.7 Histochemical GUS Analysis 49
• 2.3.8 Subcellular Localization of MRN1 49
• 2.3.9 Synthesis of (+) Marnerol 50
• 2.3.10 Quantification of Sterols, Triterpenols, and Marnerol 50
• 2.4 RESULTS 55
• 2.4.1 Isolation of a Late-flowering Mutant with Round-shaped Rosette Leaves 55
• 2.4.2 Delayed Embryo Development of the mrn1 Mutant 58
• 2.4.3 mrn1 Is a Single Recessive Mutant 60
• 2.4.4 Cell Expansion or Elongation Is Inhibited in Leaves of mrn1 62
• 2.4.5 Cell Expansion or Elongation Is Inhibited in Root and Shoot Apical Meristems of mrn1 64
• 2.4.6 Marnerol Was Detected in Transgenic Arabidopsis Overexpressing MRN1 68
• 2.4.7 The Levels of Sterols and Triterpenols Are Altered in the mrn1 Mutant 72
• 2.4.8 Changes in Membrane Integrity and Permeability Were Observed in mrn1 Mutant 75
• 2.4.9 The MRN1 Gene Is Specifically Expressed in Shoot and Root Apical Meristems 77
• 2.4.10 MRN1 Is Localized to the Endoplasmic Reticulum 79
• 2.5 DISCUSSION 81
• 2.6 REFERENCES 86
• CHAPTER 3 Bifunctional AP2/ERF Transcription Factor DARF Controls Wax and Anthocyanin Biosynthesis, Senescence, and Innate Immunity under Extended Darkness 96
• 3.1 ABSTRACT 97
• 3.2 INTRODUCTION 98
• 3.3 MATERIALS AND METHODS 103
• 3.3.1 Plant Materials and Growth Conditions 103
• 3.3.2 Construction of 35Spro:DARF Transgenic Plant 104
• 3.3.3 Construction of DARFpro:GUS Transgenic Plant 104
• 3.3.4 Construction of β-Galactosidase Assay 104
• 3.3.5 Construction of Transactivation Assay 104
• 3.3.6 Construction of β-Estradiol Inducible Plasmid 105
• 3.3.7 RNA Isolation and qRT-PCR Analysis 105
• 3.3.8 Subcellular Localization, Histological Assays and β-Galactosidase Assay 105
• 3.3.9 Staining with Toluidine Blue 106
• 3.3.10 SEM and TEM 107
• 3.3.11 Wax and Cutin Analysis 107
• 3.3.12 Microarray Assay 108
• 3.3.13 Transcriptional Activation Assay 109
• 3.3.14 ChIP Assays 109
• 3.3.15 Electrophoretic Mobility Shift Assay 110
• 3.3.16 Water Loss Assay 110
• 3.3.17 UV-B Sensitivity Assay 110
• 3.3.18 Pathogen Sensitivity Assay 111
• 3.3.19 Chlorophyll Leaching Assays and ROS Measurement 111
• 3.3.20 Measurement of Anthocyanin Content 111
• 3.4 RESULTS 118
• 3.4.1 Decreased Accumulation of Cuticular Wax Loads in Extended Darkness 118
• 3.4.2 Down-regulation of Cuticular Wax Biosynthetic Genes in Extended Darkness 120
• 3.4.3 Isolation of the DARF Gene Encoding the AP2/ERF Transcription Factor 122
• 3.4.4 DARF Is Ubiquitously Expressed in Arabidopsis 124
• 3.4.5 Structure and Subcellular Localization of DARF 126
• 3.4.6 DARF Binds to the GCC-box Elements 128
• 3.4.7 DARF Acts as a Transcriptional Activator 130
• 3.4.8 DARF Overexpression Lines and darf Mutant Exhibits Altered Cuticle Phenotypes 132
• 3.4.9 Cuticle Deposition Altered in DARF Overexpression Lines and darf Mutant 135
• 3.4.10 DARF Binds to Consensus Motif in the Promoters of Wax Biosynthetic Genes 138
• 3.4.11 darf Mutant Contributes to Drought Resistance 141
• 3.4.12 DARF Binds to Consensus Motif in the Promoters of Flavonol/Anthocyanin Biosynthetic Genes 143
• 3.4.13 UV-B Hypersensitivity of DARF Overexpression Lines 147
• 3.4.14 DARF Binds to Consensus Motif in the Promoters of Dark-/Pathogene-inducible Genes 149
• 3.4.15 darf Mutant Is Sensitive to Extended Darkness Stresses 153
• 3.4.16 darf Mutant Is Sensitive to Fungal Pathogen Infections 156
• 3.5 DISCUSSION 158
• 3.6 REFERENCES 163
• CONCLUSIONS 173
• ABSTRACT (in Korean) 175
• CURRICULUM VITAE 178