Benzoic acid metabolism in Sorbus aucuparia cell suspension cultures Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr.rer.nat.) genehmigte D i s s e r t a t i o n von Mariam Mamdoh Magdy Nashed Gaid aus Assiut / Ägypten 1. Referent: Professor Dr. Ludger Beerhues 2. Referent: apl. Professor Dr. Dirk Selmar eingereicht am: 16.06.2010 mündliche Prüfung (Disputation) am: 17.09.2010 Druckjar 2010 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakulät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Gaid MM, Sircar D, Beuerle T, Mitra A, Beerhues L. Benzaldehyde dehydrogenase from chitosan-treated Sorbus aucuparia cell cultures. J Plant Physiol 166: 1343-1349 (2009) Gaid MM, Scharnhop H, Ramadan H, Beuerle T, Beerhues L. 4-Coumarate:CoA ligase family members from elicitor-treated Sorbus aucuparia cell cultures producing benzoate- derived phytoalexins. Submitted (2010) Tagungsbeiträge Mariam M Gaid, Debabrata Sircar, Till Beuerle, Adinpunya Mitra, Ludger Beerhues; Benzoic acid metabolism in Sorbus aucuparia cell cultures (Poster). Deutsche Botanische Gesellschaft, Botanikertagung, University of Leipzig, 6-10th September, 2009 Ines Belhadj, Mariam M Gaid, Ludger Beerhues; Hyperforin biosynthesis: cDNA cloning of isobutyrophenone synthase (Poster), 58th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product research, Berlin, 29th August- 2nd September 2010 ACKNOWLEDGMENT I would like to express my special gratitude to my supervisor Professor Dr. Ludger Beerhues for providing the interesting research point and facilities to pursue my PhD study at the Institut für Pharmazeutische Biologie. His valuable supervision, guidance, enthusiasm and especially great patience have facilitated the completion of this dissertation. It is important to mention that four years ago I was feeling my way through my project. There were days when I was trying to figure out the problems, which can be quite frustrating for someone new. But you've helped me through that. Now, I look back on that time with a bit of amusement. The things that seemed so difficult then are a breeze now. I’m also thankful to Professor Dr. Benye Liu whose patience, guidance, constructive discussion, valuable ideas and support enabled me to finish this work. My great appreciation is given to Dr. Till Beuerle for his GC-MS and LC-MS analyses, analytical assistance and useful discussions. I would like to express my appreciation to Dr. Rainer Lindigkeit for much helpful advice with software problems. My warmest thanks to my colleagues of the Institute of Pharmaceutical Biology at the TU- Braunschweig, present or past, who made this study a successful experience. I especially thank Ines Rahaus for sequencing my DNA samples and her timely help. Dr. Asya Swiddan, Rawad Zodi, Andreas Müller, Iman Abd-El Rahman, Ines Belhadj, Maren Lütge and Doris Glindemann for the pleasant working environment and companionship during laboratory works. I’m so grateful to Cornelia Hüttner for her timeless help during my stay in Germany, encouragement and her presence when needed. Honestly, I shall never find words to thank her. I’m very lucky to have a senior colleague like Dr. Helge Scharnhop in our research group. It is my privilege to sincerely thank him for his continuous help in all aspects. My special thanks go to my colleague Dr. Debabrata Sircar. I really appreciate his encouragement and support. If I can ever return the favor, please let me know. I would gladly be of help. I’m grateful to my colleague Inis Winde for her help in radioisotopic assays. I acknowledge the Egyptian Government for granting me a PhD-scholarship. My deep thanks to the staff members of my department in Egypt for offering me such an opportunity to study in Germany and for their continuous encouragement. I would like to express my special thanks to my husband Hany for his support and love and to my son Kerolos for his lovely smile that can relieve any kind of tiredness. Finally, my sincere thanks are given to my family in Egypt, especially my mother Professor Dr. Sozan Tous (Department of Pharmaceutics, Assiut University, Egypt) who provided me with continuous moral support during my stay in Germany and my whole life. Contents I I. Introduction _____________________________________________________________________1 1 Sorbus aucuparia L. ____________________________________________________________1 2 Plant secondary metabolites______________________________________________________2 2.1 Phenylpropanoids ____________________________________________________________3 2.1.1 Lignin _________________________________________________________________4 2.1.2 Flavonoids ______________________________________________________________5 2.2 Phytoalexins ________________________________________________________________6 3 Biosynthesis of related secondary metabolites _______________________________________8 3.1 Benzoic acids (BAs) __________________________________________________________8 3.1.1 Biosynthesis of benzoic acids in plants ________________________________________8 3.1.2 Activation of benzoic acid _________________________________________________12 3.2 Biosynthesis of flavonoids ____________________________________________________14 3.3 Biosynthesis of polyketides other than flavonoids __________________________________15 4 Research Strategies and Objectives_______________________________________________17 II. Materials and Methods __________________________________________________________18 1 Materials ____________________________________________________________________18 1.1 Plant material ______________________________________________________________18 1.2 Chemicals _________________________________________________________________18 1.3 Nutrient media _____________________________________________________________21 1.4 Buffers and solutions ________________________________________________________23 1.5 Materials used for molecular biology ____________________________________________28 1.5.1 Host cells and Cloning vector ______________________________________________28 1.5.2 Primers ________________________________________________________________29 1.5.3 Enzymes ______________________________________________________________32 1.5.4 Kits __________________________________________________________________32 1.6 Equipments ________________________________________________________________33 2 Biochemical methods __________________________________________________________34 2.1 Stabilization of in vitro cultures ________________________________________________34 2.2 Elicitation of in vitro cultures __________________________________________________34 2.3 Enzyme extraction __________________________________________________________35 2.4 Determination of protein content _______________________________________________36 2.5 Benzaldehyde dehydrogenase (BD) assay ________________________________________36 2.6 Biphenyl synthase (BIS) assay _________________________________________________36 2.7 GC-MS analysis of BD assay __________________________________________________37 2.8 HPLC analysis of BD assay ___________________________________________________38 2.9 HPLC analysis of BIS assay ___________________________________________________40 2.10 Time course changes in BD and BIS activities ___________________________________40 2.11 Biochemical characterization of BD ___________________________________________40 2.12 p-Coumarate-CoA ligase assay _______________________________________________42 2.13 Benzoate-CoA ligase assay __________________________________________________42 2.14 Purification of enzymatic p-coumaroyl-CoA _____________________________________43 2.15 Electrospray ionization-mass spectrometry (ESI-MS) ______________________________43 2.16 Biochemical characterization of 4CL ___________________________________________44 3 Molecular biology methods _____________________________________________________45 3.1 Design of gene specific primers ________________________________________________45 3.2 Isolation of total RNA and genomic DNA ________________________________________45 3.3 Quantification of RNA and DNA concentration ___________________________________45 3.4 Reverse transcription (RT) ____________________________________________________46 3.4.1 cDNA-synthesis using SMART-RACE protocol _______________________________46 3.4.2 cDNA-synthesis using H Minus M-MuLV RT _________________________________47 3.5 Polymerase Chain Reaction (PCR) _____________________________________________47 3.5.1 Touch down PCR _______________________________________________________48 Contents II 3.5.2 Rapid amplification of cDNA ends (RACE) ___________________________________49 3.5.3 TATA-box protocol ______________________________________________________49 3.6 Agarose gel electrophoresis ___________________________________________________51 3.7 Purification of DNA from agarose gels __________________________________________51 3.8 Cloning of PCR product ______________________________________________________51 3.8.1 Cloning into pGEM-T Easy vector __________________________________________51 3.8.2 Cloning of p-coumarate-CoA ligase into expression vectors ______________________52 3.8.3 Standard restriction reaction _______________________________________________52 3.8.4