Fazeelat Karamat Laboratoire d’Agronomie et Environnement Director of thesis : Hehn Alain Co-Director of thesis:

1. functionalcharacterization of the first prenyltransferaseinvolved in the biosynthesis of coumarins, important molecules in the defense system of higher plants Fazeelat Karamat Laboratoire d’Agronomie et Environnement Director of thesis : Hehn Alain Co-Director of thesis: Bourgaud Frédéric / Olry Alexandre Séminaire RP2E - 19/01/2012 – F. Karamat

2. What are coumarin? • Implication of coumarins • Self defense system of plants (Insecticides, Bactericide and Allelochemicals) Introduction • Other implications in different industries • Pharmaceutical industry (Anti coagulant, Antitumoral, Anti-HIV, Antifungic) • Cosmetic industry ( Dye , Fragrances) • Food industry ( Flavors) Séminaire RP2E - 19/01/2012 – F. Karamat

3. Biosynthetic pathway of phenylpropanoid 4CL / HCT / C3’H… Lignin C4H/4CL PAL Flavonoid Chlorogenic Acid……. COCoA Phenylalanine Cinnamic acid p-coumaroyl coA C2’H Introduction Umbelliferone Prenyltransferase Prenyltransferase Osthenol DMS Angular form Furanocoumarins Linear form Séminaire RP2E - 19/01/2012 – F. Karamat

4. What is prenylation? The addition of an isoprene unit Prenyltransferase (PT) : enzyme that catalyze the prenylation step polyphenol Prenylated polyphenol Isoprene group Prenylated phenols in plants Introduction Euphorbiaceae Rutaceae Leguminosae Anti-poliferative activity Drug enhancement effects Anti-tyrosinase Moraceae Umbelliferae Anti-glycosidase Anti-cancer Bactericide Anti-oxidant Phytoalexin (Park et al., 2003;Stevens et al., 2000; Riberio et al., 2008) Séminaire RP2E - 19/01/2012 – F. Karamat

5. Prenylated phenols in plants Introduction Leguminosae Umbelliferae Rutaceae Celery, parsley Lime, lemon, Ruta SfN8DT Angelica EST Sequencing Séminaire RP2E - 19/01/2012 – F. Karamat

6. 1) Identification of new candidate genes Flavonoids Vitamin E Experimental strategy Flavonoids or others? Séminaire RP2E - 19/01/2012 – F. Karamat

7. Experimental strategy Done by Prof Yazaki (collaboration) Identification of new candidate genes Cloning of the genes in plasmidic expression vectors Plant Heterologous expression system Nicotiana benthamiana. Heterologous expression of the corresponding proteins Experimental strategy Functional characterization of proteins Séminaire RP2E - 19/01/2012 – F. Karamat

8. 2) Heterologous expression system The principle of the approach Promotor N-Term ORF Agrobacterium Plasmid with concerned gene Experimental strategy Protein extraction(4 days post inoculation) Séminaire RP2E - 19/01/2012 – F. Karamat (Voinnet et al., 2003; Kapila et al., 1997)

9. 2) Heterologous expression system Validation of the system in two steps 1) Test with the Jellyfish Green Fluorescent Protein (GFP) Promotor GFP N-Term Visible spectra Experimental strategy Fluorescence excitation/emission spectra -GFP +GFP Séminaire RP2E - 19/01/2012 – F. Karamat

10. 2) Heterologous expression system Validation of the system in two steps 2) Test with a membrane-bound-enzyme : the Sophora flavescens prenyltransferase ( a flavonoid specific prenyltransferase that has been characterized already) Promotor SfN8DT N-Term Infiltration of tobacco leaves 4 days post inoculation Ultra-turrax was used to prepare a homogeniezed mixture of fresh plant material. Experimental strategy Centrifugation and ultracentrifugation was done to get membrane pellets Séminaire RP2E - 19/01/2012 – F. Karamat Crude membrane extracts

11. Results Validation of the system in two steps 2) Test with an enzyme : the Sophora flavescens prenyltransferase Promotor SfN8DT N-Term With enzyme (presence of 8dN) naringenin Reaction Without enzyme Primary Results The heterologous expression system is functional Séminaire RP2E - 19/01/2012 – F. Karamat

12. Expression of 6 candidate PTs genes Promotor Putative PT ORF N-Term Isoprenic moeity Test of the 6 candidate genes PT-Lime PT-Ruta PT-Angelic PT-Lemon PT-Celery PT-Parsley Results With enzyme Without enzyme Séminaire RP2E - 19/01/2012 – F. Karamat

13. Biochemical parameters? Results Optimal pH=8.2 Km=24 +/-3 µM In agreement withalreadycharacterizedprenyltransferase (Sfn8DT, SfG6DT) Séminaire RP2E - 19/01/2012 – F. Karamat

14. Biochemical parameters? Phenylalanine Inhibition of the reaction with psoralen Umbelliferone Prenyltransferase Demetylsuberosin Osthenol Results Quantity Of DMS in nM Angelicin Psoralen Linear furanocoumarins Angular furanocoumarins Conc. Of Psoralen in µM Séminaire RP2E - 19/01/2012 – F. Karamat

15. Conclusions • In this work we developed another way to screen the activity of PT enzymes using a plant transient expression system (previously it was done in yeast) • We characterized a new enzyme involved in the biosynthetic pathway of coumarins. • It is the first prenyltransferase identified in parsley, a furanocoumarin producing plant. Phenylalanine Umbelliferone Conclusion and perspective Prenyltransferase Prenyltransferase - • We showed a potential way to regulate the production of furanocoumarins in plants thanks to the negative feedback of psoralen Demetylsuberosin Osthenol Angelicin Psoralen Angular furanocoumarins Linear furanocoumarins Séminaire RP2E - 19/01/2012 – F. Karamat

16. Project perspective • Parsley PT • In parsley • - Tissue specific expression pattern of gene ( leaves, stem, roots and flower) • - Subcellular localization ( mitochondria, endoplasmic reticulum) • In Rutagraveolens: • -Stable overexpression of the gene in order to do metabolic engineering Conclusion and perspective 2) The five other PT genes ? -Enlarged metabolic screening -Stable expression in Ruta graveolens (under way) / metabolic characterization Séminaire RP2E - 19/01/2012 – F. Karamat

17. Thank you for your attention!

18. PT- parsley is bifunctional? Umbelliferone Prenyltransferase Demetylsuberosin Osthenol Psoralen Linear furanocoumarins Without enzyme With enzyme