University of Hohenheim Institute of Plant Breeding, Seed Science and Population Genetics Plant Breeding and Biotechnology Prof. Dr. Gerd Weber Metabolic engineering of flavonoid biosynthesis in hop (Humulus lupulus L.) for enhancing the production of pharmaceutically active secondary metabolites Cumulative Doctoral Thesis in fulfillment of the requirements for the degree “Doktor der Agrarwissenschaften”(Dr.sc.agr. / Ph. D. in Agricultural Sciences) submitted to the Faculty of Agricultural Sciences by Andrés Mauricio Gatica Arias, M. Sc born in San José, Costa Rica 2012 This thesis was accepted as a doctoral dissertation in fulfillment of the requirements for the degree of “Doktor der Agrarwissenschaften” (Dr.sc.agr.) at the Faculty of Agricultural Sciences at the University of Hohenheim on 11 December 2012. Date of oral examination: 18 December 2012. Examination committee Vice-Dean and Head of the Examination committee: Prof. Dr. K. Stahr Supervisor and reviewer: Prof. Dr. G. Weber Co-reviewer: Prof. Dr. A. Pfitzner Additional examiner: Dr. habil G.M. Reustle Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdientes (DAAD) ii To my loving parents, Javier and Patricia, who supported me throughout my life iii Table of contents List of figures ....................................................................................................................................................... viii List of tables ......................................................................................................................................................... xiii List of abbreviations............................................................................................................................................... xv Summary ............................................................................................................................................................. xvii Zusammenfassung ................................................................................................................................................. xix Resumen ................................................................................................................................................................ xxi 1. Introduction ......................................................................................................................................................... 1 1.1 General information about hop ..................................................................................................................... 1 1.2 Uses of hop ................................................................................................................................................... 2 1.3 Composition of secondary metabolites in hop .............................................................................................. 3 1.3.1 Essential oils .......................................................................................................................................... 3 1.3.2 Bitter acids ............................................................................................................................................. 4 1.3.3 Prenylflavonoids .................................................................................................................................... 6 1.4 Flavonoids..................................................................................................................................................... 7 1.4.1 Structure of flavonoids .......................................................................................................................... 7 1.4.2 Functions of flavonoids ......................................................................................................................... 7 1.4.3 Biosynthetic pathway of flavonoid biosynthesis ................................................................................... 9 1.5 Regulation of flavonoid biosynthesis .......................................................................................................... 11 1.5.1 MYB transcription factors ................................................................................................................... 11 1.5.2 Transcription factors in hop ................................................................................................................. 12 1.6 Metabolic engineering of flavonoids .......................................................................................................... 13 1.7 Objectives of the thesis ............................................................................................................................... 15 1.8 Publications................................................................................................................................................. 15 2. Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L. ..................................................................................................................................... 17 2.1 Abstract ....................................................................................................................................................... 17 2.2 Introduction................................................................................................................................................. 18 2.3 Material and methods.................................................................................................................................. 19 2.3.1 Plant transformation, regeneration and acclimatization ....................................................................... 19 2.3.2 Detection of transgenic plants ............................................................................................................. 19 2.3.3 RNA isolation and RT-PCR ................................................................................................................ 20 2.3.4 Gene expression analysis of PAP1/AtMYB75 ...................................................................................... 21 2.3.5 Extraction procedure and sample preparation...................................................................................... 22 2.3.6 LC-MS analysis ................................................................................................................................... 22 iv 2.3.7 Anthocyanins quantification ................................................................................................................ 22 2.4 Results ........................................................................................................................................................ 23 2.4.1 PCR detection of transformed plants ................................................................................................... 23 2.4.2 Analysis of transgene expression by RT-PCR ..................................................................................... 23 2.4.3 Quantitative analysis of transgene expression by real time PCR ......................................................... 24 2.4.4 Morphology of transgenic plants and chemical analysis of secondary metabolites............................. 25 2.5 Discussion ................................................................................................................................................... 29 2.6 Acknowledgements ..................................................................................................................................... 31 2.7 References................................................................................................................................................... 32 3. The transcription factor AtMYB75/PAP1 regulates the expression of flavonoid biosynthesis genes in transgenic hop (Humulus lupulus L.) ............................................................................................................... 34 3.1 Abstract ....................................................................................................................................................... 34 3.2 Introduction................................................................................................................................................. 35 3.3 Materials and methods ................................................................................................................................ 36 3.3.1 Plant material ....................................................................................................................................... 36 3.3.2 Primer design and assessment of specificity ........................................................................................ 36 3.3.3 Estimation of AtMYB75/PAP1 copy number ....................................................................................... 37 3.3.4 Expression analysis of flavonoid biosynthesis genes .......................................................................... 38 3.3.5 Chemometrics analysis ........................................................................................................................ 39 3.4 Results and discussion ................................................................................................................................ 40 3.4.1Phenotypic characterization of AtMYB75/PAP1 transgenic plants ......................................................