.......................................................................................................................................... 3 Introduction: .................................................................................................................................... 5 The polyphenol oxidase enzyme ................................................................................................. 5 Role of polyphenol oxidase in tyrosine and dopamine metabolism ............................................ 7 Role of polyphenol oxidase in esculetin synthesis ...................................................................... 9 Analysis of PPO activity and function: ..................................................................................... 11 Objectives: .................................................................................................................................... 12 Materials and methods: ................................................................................................................. 14 Protein extraction ...................................................................................................................... 14 Substrate specificity and enzyme kinetic assays ....................................................................... 14 Sequence alignment and comparison of catechol oxidases and cresolases ............................... 15 Analyzing PPO genes in California poppy ............................................................................... 15 Maintenance of California poppy cell cultures ......................................................................... 16 Cell culture growth rate ............................................................................................................. 16 Metabolite extraction from poppy cell cultures ........................................................................ 17 HPLC analysis of BIA content .................................................................................................. 18 Data analysis ............................................................................................................................. 18 Targeted metabolite profiling of dopamine and esculetin in red clover ................................... 19 Results: .......................................................................................................................................... 20 Plant PPO enzyme kinetics ....................................................................................................... 20 Targeted metabolite profiling of dopamine and esculetin in wild-type and PPO-silenced red clover ......................................................................................................................................... 29 Metabolic effects of inhibiting PPO in California poppy ......................................................... 30 Discussion: .................................................................................................................................... 40 Future directions: .......................................................................................................................... 44 References Cited: .......................................................................................................................... 45 Appendix A: .................................................................................................................................. 52