Role of MYB94 and MYB96 in cuticular wax biosynthesis in Arabidopsis thaliana and functional application of MYB96 in Camelina sativa = 애기장대 큐티클 왁스 생합성에서 MYB94와 MYB96의 역할 및 카멜리나에 MYB96의 기능 적용

다국어 초록 (Multilingual Abstract)

The aerial parts of plants are covered with a cuticle, a hydrophobic layer consisting of cutin polyester and cuticular waxes that protects them from various environmental stresses. Cuticular waxes mainly comprise very long chain fatty acids and their derivatives such as aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax esters. The major function of cuticular waxes is to control non-stomatal water loss and gas exchange. Cuticular waxes are also important raw materials for the production of lubricants, adhesives, cosmetics, and biofuels. Cuticular wax deposition is influenced by environmental changes. However, the regulatory mechanism of cuticular wax biosynthesis in response to environmental stresses is largely unknown. In the present study, a novel MYB94 transcription factor that activates Arabidopsis cuticular wax biosynthesis was identified. MYB94 is abundantly expressed in aerial organs and highly expressed in the stem epidermis than within the stem. The expression of the MYB94 was increased in response to drought, ABA, NaCl and mannitol treatments. MYB94 harbors the transcriptional activation domain in the C-terminal region. Fluorescent signals from MYB94:enhanced yellow fluorescent protein (eYFP) were observed in the nucleus of tobacco epidermal cells and in transgenic Arabidopsis roots. The total wax loads increased by approximately 2-fold in the leaves of the MYB94-overexpressing (MYB94 OX) lines, as compared with those of the wild type. MYB94 activates the expression of WSD1, KCS2/DAISY, CER2, FAR3 and ECR genes by directly binding to their gene promoters. The cuticular transpiration occurred more slowly in the leaves of MYB94 OX lines compared with the wild type, under drought stress conditions. These results indicate that a R2R3-type MYB94 transcription factor activates Arabidopsis cuticular wax biosynthesis and might be important in plant response to environmental stresses such as drought stress. In the phylogenetic tree of Arabidopsis R2R3-type MYB transcription factors, MYB94 is most closely related to MYB96. MYB96 has a transcriptional activation domain in the C-terminal region that is similar to MYB94. Both MYB96 and MYB94 were abundantly expressed in the aerial organs of Arabidopsis, and were significantly induced at the similar time by water deficiency. MYB94 complemented the wax-deficient phenotype of myb96. The magnitude of decrease in total wax loads in the myb96 myb94 double mutant was almost equal to the sum of the reduced wax loads in myb96 and myb94 mutants under well-watered and drought-stress conditions. The cuticular transpiration occurred more rapidly in myb96 myb94 leaves than in single mutant myb96 or myb94 leaves. The expression levels of wax biosynthetic genes that are directly regulated by MYB96 and/or MYB94 decreased in myb96 and myb94, and were further decreased in myb96 myb94 double mutants relative to wild type. MYB96 and MYB94 interact with the same consensus motifs in the promoters of their target genes. These data show that the MYB96 and MYB94 transcription factors redundantly and additively function in cuticular wax biosynthesis, which might be an efficient and adaptive mechanism in plants in response to drought. Then, the Arabidopsis MYB96 gene was overexpressed in Camelina under the control of the CaMV35S promoter. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited normal growth and development and enhanced tolerance to drought. Deposition of epicuticular wax crystals and total wax loads significantly increased on the surfaces of transgenic leaves compared with that of non-transgenic plants. The levels of alkanes and primary alcohols prominently increased in transgenic Camelina plants relative to non-transgenic plants. Cuticular transpiration occurred more slowly in transgenic leaves than that in non-transgenic plants. Genome-wide identification of Camelina wax biosynthetic genes enabled us to determine that the expression levels of CsKCS2, CsKCS6, CsKCR1-1, CsKCR1-2, CsECR, and CsMAH1 were approximately 2- to 7-fold higher in transgenic Camelina leaves than those in non-transgenic leaves. These results indicate that MYB96-mediated transcriptional regulation of wax biosynthetic genes is an approach applicable to generating drought-resistant transgenic crops.

목차 (Table of Contents)

• CONTENTS
• LIST OF TABLES AND FIGURES
• ABBREVIATIONS
• ABSTRACT (in English) 1
• CHAPTER 1 General Introduction 5
• 1.1 Cuticular wax amounts and compositions in Arabidopsis and crop plants 9
• 1.2 Cuticular wax biosynthesis in Arabidopsis 13
• 1.3 Cuticular wax transport in Arabidopsis 21
• 1.4 Regulation of cuticular wax biosynthesis in Arabidopsis 23
• 1.5 Cuticular wax biosynthesis and transport in crop plants 28
• 1.6 Regulation of cuticular wax biosynthesis in crop plants 32
• 1.7 Perspectives 34
• 1.8 The purpose and contents of this study 36
• CHAPTER 2 Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in Arabidopsis 37
• 2.1 ABSTRACT 38
• 2.2 INTRODUCTION 39
• 2.3 MATERIALS AND METHODS 43
• 2.3.1 Plant materials and growth conditions 43
• 2.3.2 Construction of binary vectors and Arabidopsis transformation 43
• 2.3.3 Transactivation assay of MYB94 in yeast 44
• 2.3.4 Subcellular localization of MYB94:enhanced yellow fluorescent protein 45
• 2.3.5 Gene expression analysis 45
• 2.3.6 GUS staining and microscopy 46
• 2.3.7 Scanning electron microscopy (SEM) analysis 47
• 2.3.8 Cuticular wax analysis 47
• 2.3.9 Cutin polyester analysis 48
• 2.3.10 Microarray analysis 48
• 2.3.11 Transcriptional activation assay 49
• 2.3.12 Electrophoretic mobility shift assay (EMSA) 50
• 2.3.13 Cuticular transpiration assay 50
• 2.4 RESULTS 55
• 2.4.1 Isolation of MYB94 gene encoding R2R3-type MYB transcription factor 55
• 2.4.2 MYB94 contains a transcriptional activation domain in its C-terminal region and the fluorescent MYB94:eYFP is localized to the nucleus 58
• 2.4.3 Spatial and temporal expression patterns of MYB94 in various Arabidopsis organs and in seedlings after treatments with ABA, drought, salt and osmotic stress 62
• 2.4.4 The amounts of cuticular waxes increased in the leaves of transgenic Arabidopsis plants overexpressing MYB94 66
• 2.4.5 The expression of genes involved in cuticular wax biosynthesis was up-regulated in leaves of transgenic Arabidopsis plants overexpressing MYB94 72
• 2.4.6 MYB94 activates the expression of wax biosynthetic genes via direct binding to their promoters 76
• 2.4.7 Cuticular transpiration occurred more slowly in the leaves of the MYB94 OX lines compared with those of the wild type 80
• 2.5 DISCUSSION 82
• CHAPTER 3 MYB96 and MYB94 redundantly and additively function in Arabidopsis cuticular wax biosynthesis 87
• 3.1 ABSTRACT 88
• 3.2 INTRODUCTION 89
• 3.3 MATERIALS AND METHODS 94
• 3.3.1 Plant materials and growth conditions 94
• 3.3.2 Isolation of myb94 single and myb96 myb94 double mutants, and complementation of myb96 and myb94 mutants 94
• 3.3.3 RT-PCR and quantitative RT-PCR (qRT-PCR) analyses 95
• 3.3.4 Transactivation assay of MYB96 in yeast 96
• 3.3.5 Cuticular wax analysis under normal and drought stress conditions 96
• 3.3.6 Cuticular transpiration and chlorophyll leaching assays 96
• 3.3.7 Chromatin immunoprecipitation (ChIP) assay 97
• 3.3.8 Bimolecular fluorescence complementation (BiFC) assay 97
• 3.3.9 Firefly luciferase complementation imaging (LCI) assay 98
• 3.4 RESULTS 102
• 3.4.1 Comparison of MYB96 and MYB94 protein structures and abiotic stress-induced expression of MYB96 and MYB94 102
• 3.4.2 The myb96 wax-deficient phenotype was rescued by the expression of MYB94 under well-watered conditions 106
• 3.4.3 Isolation of T-DNA-inserted myb94 knockout mutant and generation of myb96 myb94 double mutant 109
• 3.4.4 myb94 exhibited a wax-deficient phenotype, which was restored to wild type by the expression of MYB94 under the control of the MYB94 promoter 113
• 3.4.5 MYB96 and MYB94 additively function in cuticular wax biosynthesis under both well-watered and drought stress conditions 116
• 3.4.6 The myb96 myb94 double mutant has a more permeable cuticle than each single myb96 or myb94 mutant 120
• 3.4.7 Expression of wax biosynthetic genes, which are directly regulated by MYB96 and/or MYB94 in wild type, myb96, myb94, and myb96 myb94 under well-watered and drought-stress conditions 122
• 3.4.8 Both MYB96 and MYB94 bind to the same cis-elements in the promoters of wax biosynthetic genes 124
• 3.5 DISCUSSION 126
• CHAPTER 4 Overexpression of Arabidopsis MYB96 confers drought resistance in Camelina sativa via cuticular wax accumulation 133
• 4.1 ABSTRACT 134
• 4.2 INTRODUCTION 135
• 4.3 MATERIALS AND METHODS 140
• 4.3.1 Plant materials and growth conditions 140
• 4.3.2 Camelina transformation and selection of Camelina transgenic plants 140
• 4.3.3 Genomic DNA isolation, RNA isolation, and PCR analysis 141
• 4.3.4 Drought stress treatments 141
• 4.3.5 Stomatal density analysis 142
• 4.3.6 Cuticular transpiration and chlorophyll leaching assays 142
• 4.3.7 SEM analysis 142
• 4.3.8 Cuticular wax analysis in Camelina plants 143
• 4.3.9 Accession numbers 143
• 4.4 RESULTS 145
• 4.4.1 Expression of Arabidopsis MYB96 in Camelina 145
• 4.4.2 Transgenic Camelina plants overexpressing Arabidopsis MYB96 confer increased resistance to drought 149
• 4.4.3 An increase of cuticular wax deposition in leaves of transgenic Camelina plant overexpressing Arabidopsis MYB96 152
• 4.4.4 Transcript levels of Camelina cuticular wax biosynthetic genes were up-regulated in transgenic Camelina plants 156
• 4.5 DISCUSSION 160
• CONCLUSIONS 168
• REFERENCES 172
• ABSTRACT (in Korean) 196
• CURRICULUM VITAE 200