Molecular aspects in the biosynthesis of diterpene secondary metabolites in stevia rebaudiana and stevia ovata

Table of contents Page Acknowledgements i List of figures vii List of tables ix List of abbreviations xi Introduction 1 Chapter 1: Literature review 5 1.1 Stevia: history of discovery 5 1.2 General features of Stevia rebaudiana and Stevia ovata 6 1.3 Sweet glycosides in Stevia rebaudiana 8 1.3.1 Stevioside 10 1.3.1.1 Pharmaceutical uses of stevioside 10 1.3.1.2 Safety of stevioside 11 1.3.2 Rebaudioside A 12 1.3.2.1 Safety of rebaudioside A 12 1.3.3 Other steviol glycosides 13 1.3.4 Radical scavenging activity of steviol glycosides 13 1.4 Metabolic flux to gibberellins and steviol synthesis pathway 14 1.4.1 Isopentenyl pyrophosphate 14 1.4.2 Geranylgeranyl pyrophospate 18 1.4.3 Ent-kaurenoic acid as a common precursor for gibberellins and steviol 18 1.5 Different gene families involved in the biosynthetic pathway of steviol 20 1.5.1 Terpene cyclases 20 1.5.1.1 Ent-copalyl pyrophosphate synthase 21 1.5.1.2 Ent-kaurene synthase 22 1.5.2 Cytochrome P450 genes 24 1.5.2.1 From ent-kaurene to ent-kaurenoic acid 24 1.5.2.2 The branch point 26 1.5.3 Down-stream genes: UDP-dependent glycosyl transferases 26 1.5.3.1 Classification of SrUDP-dependent glycosyltransferases 28 1.6 Non-sweet glycosides in Stevia ovata 30 Chapter 2: Materials and Methods 33 2.1 Extraction and purification of ent-kaurenoic acid 33 2.1.1 Extraction of the plant material 33 2.1.2 Chromatographic separation and identification of ent-kaurenoic acid 33 2.1.3 Purification of ent-kaurenoic acid 34 2.1.4 Mass spectrometric analysis 34 2.1.5 Nuclear magnetic resonance (NMR) analysis 34 2.2 Cloning of a cytochrome P450 gene as possible candidate for KAHent 36 2.2.1 cDNA cloning and analysis 36 2.2.2 DNA extraction 39 2.3 Transcription pattern of the genes involved in the biosynthesis of steviol glycosides 40 2.3.1 Plant material and growth conditions 40 2.3.2 Steviol glycoside quantification 40 2.3.3 Extraction of RNA and cDNA synthesis 41 2.3.4 Choosing the endogenous control (the housekeeping gene) 41 2.3.5 Primer design 42 2.3.6 Quantification using RT-qPCR 43 2.3.7 Statistical analysis 44 2.4 Molecular analysis in Stevia ovata 44 2.4.1 Growth conditions, plant material and RNA extraction 44 2.4.2 RT-PCR and PCR experiments and cloning 44 2.4.3 RT-qPCR of SoCPSent transcription 50 2.4.4 Gene expression and activity analysis 50 2.4.4.1 Gateway expression system 50 2.4.4.2 Functional assay 53 2.4.4.3 Analysis of geranylgeraniol and copalol 53 Chapter 3: Extraction and purification of ent-kaurenoic acid 55 3.1 Introduction 55 3.2 Results 55 3.2.1 Extraction of ent-kaurenoic acid and preliminary LC-MS analysis 55 3.2.2 Purification of ent-kaurenoic acid 59 3.2.3 Characterization of compounds by EI-MS 61 3.2.3 Characterization of compounds by NMR 66 3.3 Discussion 67 Chapter 4: C-13 hydroxylation of ent-kaurenoic acid to steviol 69 4.1 Introduction 69 4.2 Results 69 4.2.1 cDNA cloning and analysis 69 4.2.2 Detection of the gap at the genomic level 73 4.2.3 Cloning of the whole gene from gDNA 74 4.3 Discussion 75 Chapter 5: Regulation of steviol glycoside biosynthesis by daylength and light 83 5.1 Introduction 83 5.2 Results 83 5.2.1 Steviol glycoside accumulation 83 5.2.2 Analysis of the transcription of the genes 86 5.2.2.1 Transcription pattern of up-stream genes 86 5.2.2.2 Transcription pattern of down-stream genes 94 5.2.3 Statistical analysis 100 5.3 Discussion 103 Chapter 6: Early steps in regulation of the biosynthesis of paniculosides in Stevia ovata 109 6.1 Introduction 109 6.2 Results 109 6.2.1 cDNA cloning and analysis 109 6.2.2 RT-qPCR anaylsis of SoCPSent 111 6.3 Discussion 112 Chapter 7: General conclusion and perspectives 119 Summary 123 Samenvatting 127 References 131 List of publications 153nrpages: 167status: publishe