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Assessment of Transgenic Approaches to Increase Lycopene Content in Ripe Tomato Fruits

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Assessment of Transgenic Approaches to Increase Lycopene Content in Ripe Tomato Fruits ASSESSMENT OF TRANSGENIC APPROACHES TO INCREASE LYCOPENE CONTENT IN RIPE TOMATO FRUITS Thesis submitted for the degree of “Doctor of Philosophy” By Kolotilin Igor Submitted to the Senate of the Hebrew University April 2008 This work was carried out under the supervision of Dr. Ilan Levin, Agricultural Research Organization; Prof. Shahal Abbo, the Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem. 2 CONTENTS ABBREVIATIONS………………………………………………………….…………….……. 6 ABSTRACT……………………………………………………………………………….……. 8 BACKGROUND ………………………………………………………………………...……... 10 Carotenoids, their biological roles and health-promoting properties……………………...…… 10 Plastid non-mevalonate pathway for isoprenoid biosynthesis and its regulation…..….………... 12 Carotenoid biosynthesis pathway and its regulation …..………….............................................. 15 Genetic engineering of the carotenoid synthesis pathway in tomato fruits...……….…..……….. 17 Genetic engineering of the carotenoid content in other crops……............................................... 18 Light signal transduction (photomorphogenesis)…………….……….......................................... 20 Arabidopsis thaliana det1 mutants and extragenic suppressors of det1 phenotype………………………………………….………………………... 23 The high pigment tomato mutations…………………….………………………………….......... 24 Engineering carotenoid content through modulation of light signal transduction pathway…………………………………………..………………… 27 Other molecular approaches affecting carotenoid content in tomato……….………….………. 28 RESEARCH HYPOTHESES AND SPECIFIC OBJECTIVES……………………….......... 30 Research hypothesis I………………………………………..………………………….……….. 30 Research hypothesis II……………………………………………….…………………….……. 30 Research hypothesis III………………………………………………………………………….. 31 The major and the specific objectives of this study………………..……………………….……. 31 MATERIALS AND METHODS……………………………………..…………….…………. 32 Generation and characterization of transgenic plants overexpressing Arabidopsis thaliana (A. thaliana) ted3 gene in tomato……………………………………….. 32 Cloning of the TED3 gene of A. thaliana……………………………..……….………………… 32 Site-directed mutagenesis of the TED3 gene…………………………..…...…………….……… 32 Cloning the promoter of the ribulose-l,5-bisphosphate carboxylase/oxygenase (RbcS) small subunit 3C ………………………….….………………… 32 Construction of binary vectors for plant transformation……………………………..…………. 33 Transformation of tomato plants and identification of transgenics……….................….………. 34 Growth rate analysis of the transgenic plants overexpressing ted3……………………………... 34 3 Seedling analysis to examine reversion of the mutant hp-2j phenotype by ted3……………………………………………………………… 35 Transcriptional profiling of fruits harvested from hp-2dg and ySAMdc-overexpressing tomato plants ………………………… 35 Plant material and experimental design…………………………………..…………….………. 35 RNA extraction and amplification………………………………………….................…………. 36 Microarray experiment analysis………………………………………….……………..………. 36 Real-time PCR reactions………………………………………………….…………..…………. 38 Assignment of genes regulated in fruits overexpressing ySAMdc into specific cellular metabolic pathways……………………………………………………….. 39 Ontological analysis of regulated genes in ySAMdc-overexpressing tomato fruits …………….. 39 Microscopy and image analyses……………………………………………………...…………. 39 Carotenoids extraction and quantitation………………………………….….………………….. 40 Chlorophyll extraction………………………………………………………………..…………. 40 Soluble protein extraction…………………………………………………………….…………. 41 Statistical analyses of additional quantitative data………………………………..……………. 41 RESULTS……………………………………………………………………………...………... 42 Characterization of transgenic tomato plants overexpressing A. thaliana ted3 gene.......................................................................................... 42 Generation of transgenic plants …………………………………………..………….…………. 42 Growth rate analysis of the transgenic ted3-overexpressing plants………………….…………. 42 Expression analysis of ted3 in transgenic plants overexpressing ted3 and their fruit carotenoid content……………….….................................... 43 Carotenoid content in fruits harvested from hp-2dg mutant and ySAMdc-overexpressing tomatoes and their crosses…………………………………….. 44 Characterization of the hp-2dg tomato mutant........................................................................... 46 Carotenoids profiling of fruit pericap of hp-2dg and hp-2j tomato mutants ……………….……. 46 General transcriptional alterations in pericarp tissue of hp-2dg mutant fruits .……………………………………………………..... 47 Changes in transcription of the MEP pathway and carotenogenesis genes, observed in fruits of the hp-2dg mutant…………………………….. 49 Major transcriptional effects of the hp-2dg mutation…………………………………………….. 50 Regulation of stress-related genes in the hp-2dg tomato mutants………………………………... 51 4 Microscopy studies of the tomato hp-2dg mutant fruit………………………………….………... 52 Regulation of genes related to chromoplast biogenesis in hp-2dg mutant fruits………………… 56 Soluble protein assessment in pericarp of the hp-2dg and the hp-2j mutants……………………. 57 Characterization of tomatoes overexpressing ySAMdc (PAs-accumulating tomatoes)......... 58 Analysis of ySAMdc transcription at three stages of fruit development…………………………. 58 General transcriptional alterations in pericarp tissue of tomatoes overexpressing ySAMdc ……………………………………………………….…… 59 Ontology analysis of regulated genes in tomatoes overexpressing ySAMdc ……………………. 61 Assignment of the regulated genes onto specific cellular metabolic pathways……………………………………………..………….…………... 63 Transcriptional regulation of ethylene-related genes in ySAMdc-overexpressing tomatoes……………………………………………….……... 67 Transcriptional regulation of genes related to photorespiration in ySAMdc-overexpressing tomatoes……………………..………………………….…………... 68 DISCUSSION………………………………………………………..………………...………... 70 Analysis of tomato plants overexpressing the A. thaliana ted3 gene …….………...……....... 70 Characterization of photomorphogenic hp tomato mutants……...……….……...…………. 73 Characterization of transgenic plants overexpressing ySAMdc............................................... 79 Combining the hp-2dg mutation with ySAMdc transgene…………...…................................... 87 CONCLUSION…………………………………………………………………….......……….. 89 Tomato plants overexpressing A. thaliana ted3 gene…………………………..........……….. 89 The tomato hp-2dg mutant ……………………………………………………………….......… 89 Transgenic tomatoes overexpressing ySAMdc………………………………………………... 90 The combined hp-2dg/ySAMdc genotype………………………………………………………. 90 REFERENCES …………………………………………………..…………………………….. 92 SUPPLEMENTAL MATERIALS ……………………………………………………………. 112 ACKNOWLEDGMENTS............................................................................................................ 131 5 ABBREVIATIONS ABA = Abscisic Acid ACC = 1-Aminocyclopropane-1-Carboxylic Acid ACS = ACC synthase bHLH = Basic-Helix-Loop-Helix structural motif bZIP = Basic Leucine Zipper Domain CGS = Cystathionine g-Synthase CHY = β-CAROTENE HYDROXYLASE CMK = 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase CMS = MEP CYTIDYLYLTRANSFERASE COP1 = CONSTITUTIVELY PHOTOMORPHOGENIC 1 COP10 = CONSTITUTIVELY PHOTOMORPHOGENIC 10 CRTISO = CAROTENOID ISOMERASE CRY = Cryptochrome CSN = COP9 SIGNALOSOME Cv = Cultivar DDB1 = UV DAMAGED DNA BINDING PROTEIN 1 DET1 = DE-ETIOLATED1 DMAPP = Dimethylallyl Diphosphate DXR = DEOXYXYLULOSE 5-PHOSPHATE REDUCTOISOMERASE DXS = DEOXYXYLULOSE 5-PHOSPHATE SYNTHASE EMS = Ethyl Methanesulfonate (C3H8O3S) FUS = Fusca FW = Fresh Weight g = gram G = Gravity Force GDS = GERANYL DIPHOSPHATE SYNTHASE GGPP = GERANYLGERANYL PYROPHOSPHATE GGPS = GERANYLGERANYL PYROPHOSPHATE SYNTHASE GGR = GERANYLGERANYL REDUCTASE GMO = Genetically-Modified Organism EST = Expressed Sequence Tag HDR = HYDROXYMETHYBUTENYL DIPHOSPHATE REDUCTASE 6 HDS = HYDROXYMETHYBUTENYL DIPHOSPHATE SYNTHASE HFR1 = LONG HYPOCOTYL IN FAR-RED 1 HP = HIGH PIGMENT HPLC = High-Performance Liquid Chromatography HY5 = LONG HYPOCOTYL 5 IDI = ISOPENTENYL DIPHOSPHATE ISOMERASE IPP = Isopentenyl Diphosphate LCY = LYCOPENE CYCLASE LDL = Low Density Lipoprotein MECPP = 2-C-methyl-D-erythritol 2,4-cyclodiphosphate MEP = Methylerythritol Phosphate mm = millimeter MSR = Methionine Sulfoxide Reductase NCED = nine-cis-EPOXYCAROTENOID DIOXYGENASE ng = nano-gram PAs = Polyamines PCR = Polymerase-Chain Reaction PDS = PHYTOENE DESATURASE PHY = PHYTOCHROME PIF3 = PHYTOCHROME-INTERACTING FACTOR3 PHOT = Phototropin ppm = parts per million (1/1000000) PSY = PHYTOENE SYNTHASE QRT-PCR = Quantitative RT-PCR RT = Reverse Transcription RubisCO = Ribulose 1,5-bisphosphate Carboxylase/Oxygenase SAM1 = S-Adenosyl-L-Methionine Synthase 1 TS = Threonine Synthase UV = Ultra-Violet ySAMdc = yeast S-ADENOSYLMETHIONINE DECARBOXYLASE ZDS = ξ-CAROTENE DESATURASE ZEP = ZEAXANTHIN EPOXIDASE µg = microgram 7 ABSTRACT Background Tomatoes (Solanum lycopersicum) are an important component of human diet worldwide, consumed both fresh and processed. Tomatoes provide primarily taste and color to our diet, but can also serve as a good source of vitamins, antioxidants, minerals and fibers. These phytonutrients are known as "functional metabolites" that are thought to promote or a least maintain good health. Efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids, in tomato fruits. Previous studies show the complexity of the molecular mechanisms involved in carotenoid synthesis/sequestration, which underline limitations in increasing the carotenoid content
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