INTERFERENCE BETWEEN GERBERIN/PARASORBOSIDE AND CAROTENOID BIOSYNTHESIS Cuong Xuan Nguyen Master’s thesis University of Helsinki Department of Applied Biology Plant Production Science / Plant Breeding May 2012 HELSINGIN YLIOPISTO HELSINGFORS UNIVERSITET UNIVERSITY OF HELSINKI Tiedekunta/Osasto Fakultet/Sektion Faculty Laitos Institution Department Faculty of Agriculture and Forestry Department of Agricultural Sciences Tekijä Författare Autho rCuong Xuan Nguyen Työn nimi Arbetets titel Title Interference between gerberin/parasorboside and carotenoid biosynthesis Oppiaine Läroämne Subject Plant Breeding Työn laji Arbetets art Level Aika Datum Month and year Sivumäärä Sidoantal Number of pages Master’s Thesis May 2012 59 pages Tiivistelmä Referat Abstract Phytoene desaturase (PDS) plays a key role in the carotenoid biosynthesis in plants. Knocked-down the expression of PDS gene by virus induced gene silencing (VIGS) shows the photobleached phenotype in infected plants so that it has been used as a marker or a long time in VIGS systems with range of plant species. Tobacco rattle virus (TRV) based VIGS system, which uses PDS as the visual marker has been successfully applied and showed the white phenotype in Gerbera hybrida. However, 2-pyrone synthase (PS) gene, which encodes the first enzyme in gerebrin/parasorboside biosynthesis, is significantly reduced in these infected VIGS albino sectors. The transcription level of 2PS gene was also strongly suppressed in leaves treated with photobleaching herbicide, Norflurazon (NF), which inhibits the activity of PDS. Thus, down-regulation of 2PS gene in photobleaching sectors is caused by silencing PDS gene rather than by reacting of gerbera to TRV in VIGS treatment. Interestingly, expression of 2PS in transgenic tobacco (Nicotina tabacum SR1) causes photooxidative bleaching of the leaves. The reduction of ȕ-carotene in white leaves which analyzed by thin layer chromatograph (TLC) is the main reason; however, the interference between gerberin/parasorboside and carotenoid biosynthesis in these transgenic plants is still unclear. To overcome the effect of overexpression 2PS gene, exogenous mevalonic acid lactone (MAL) could be applied to partially rescue this transgenic phenotype at the seedling stages. Avainsanat Nyckelord Keywords VIGS, 2 pyrone synthase, phytoene desaturase, gerbera, norflurazon Säilytyspaikka Förvaringsställe Where deposited Department of Agricultural Sciences and Viikki Campus Library Muita tietoja Övriga uppgifter Further information Supervisor: Professor Teemu Teeri Table of Contents ABBREVIATIONS ...................................................................................................... 7 1 INTRODUCTION ..................................................................................................... 8 1.1 Terpenoids/Isoprenoids ....................................................................................... 8 1.1.1 Biosynthesis of plant isoprenoids .................................................................. 9 1.1.2 Plant carotenoids ........................................................................................ 12 1.2 Phenolic compounds ......................................................................................... 15 1.2.1 Plant polyketide biosynthesis ...................................................................... 16 1.2.2 Gerbera 2-pyrone synthase ......................................................................... 16 1.3 Virus induced gene silencing (VIGS), an efficient reverse genetic tool .............. 18 1.3.1 Molecular mechanism of VIGS................................................................... 18 1.3.2 Tobacco rattle virus (TRV), a successful VIGS vector................................ 19 2 OBJECTIVES .......................................................................................................... 21 3 MATERIALS AND METHODS.............................................................................. 22 3.1 Plant materials ................................................................................................... 22 3.2 Chemical treatments .......................................................................................... 23 3.3 VIGS treatment ................................................................................................. 23 3.4 RNA extraction and cDNA synthesis ................................................................. 24 3.5 Quantitative real-time PCR................................................................................ 26 3.6 Carotenoid extraction and thin layer chromatography ........................................ 27 4 RESULTS ................................................................................................................ 28 4.1 Photobleached phenotypes were observed in VIGS and NF treatment ............... 28 4.2 PDS-VIGS and Norflurazon-treated plants had reduced ȕ-carotene and accumulated phytoene ............................................................................................. 30 4.3 The accumulation of phytoene on VIGS treated plants, but not on NF treated plants, was caused by reduced expression levels of PDS ......................................... 30 4.4 Down regulation of G2PS in white sectors of VIGS and NF treated gerbera plants ............................................................................................................................... 31 4.5 Detection of TRV in VIGS-treated gerbera ........................................................ 33 4.6 Over-expression G2PS gene in tobacco caused a phototbleached phenotype, and reduction of ȕ-carotene, but not accumulation of phytoene ...................................... 34 4.7 TAL inhibited germination and growth of tobacco seedlings invitro .................. 36 4.8 Exogenous MAL rescued the tobacco over-expressing 2PS invitro conditions, but BR not..................................................................................................................... 36 5 DISCUSSION .......................................................................................................... 40 6 CONCLUSIONS ...................................................................................................... 45 7 ACKNOWLEDGEMENTS...................................................................................... 46 8 REFERENCES ........................................................................................................ 47 9 APPENDICES ......................................................................................................... 57 Appendix 1: Growth media used for rooting of Gerbera hybrida and sowing of tobacco.................................................................................................................... 57 Appendix 2: Chemicals used for rescue and toxic treatment .................................... 57 Appendix 3 Agarose gel for checking the quality of RNA ...................................... 58 Appendix 4: Details and sequence of primers used for qPCR assays ........................ 59 ABBREVIATIONS 2PS 2-pyrone synthase BR epibrassinolide CoA coenzyme A DMAPP dimethyllallyl diphosphate GAP D-glyceraldehyde-3-phosphate IPP isopentenyl diphosphate MAL mevalonic acid lactone MEP 2-C-methylerythritol 4-phosphate MES 2-(N-morpholino) ethanesulfonic acid MVA mevalonic acid NF norflurazon PDS phytoene desaturase PKS polyketide synthase RT room temperature TAL triacetolactone TLC thin-layer chromatography TRV Tobacco rattle virus VIGS virus induced gene silencing 8 1 INTRODUCTION While primary metabolites are essential for all living cell types, secondary metabolites were suspected as the waste products of plant cells in the 1950s (Hartmann 2007). Nowadays, these compounds are proven to play a key role in maintaining the ¿tness of plants in natural environment such as defense against herbivores, pathogens, competing with other plants for light, water, and nutrients or attracting insects or animal for pollinating or seed dispersing. Besides playing a signal for communication between plants and biotic environment, plant secondary metabolites also protect plants against UV light or other physical stresses (Wink 2010). Not only important for plants themselves, plant secondary metabolites have been being utilized by human for a long time as a source of medicines, dyes, insecticides, flavors and fragrances. There are nearly 200,000 plant secondary metabolites (Dixon & Strack 2003) which have enormous diversity and specificity among plant species, organs, tissues, cells or compartments of cell (Yonekura-Sakakibara & Saito 2009, Wink 2010 ). In contrast to the range of functional and structural diversities, these compounds could be divided into three main groups: phenolic, terpenoids/isoprenoids, and nitrogen, or sulfur containing compounds according to their biosynthetic origins (Aharoni & Galili 2011). Glycolysis, the Krebs cycle, and the shikimate pathway are the main primary metabolic pathways that supply precursors for secondary metabolisms. The knowledge of biosynthesis and regulation of plant secondary metabolisms is, however, still poorly understood. Thus, a better understanding of the regulatory and metabolic pathways will increase the efficiency of useful plant productions in fields such as molecular farming, functional food, and plant resistance. 1.1 Terpenoids/Isoprenoids Isoprenoids are the largest group of plant secondary metabolites, which contain