ABSTRACT ZENG, HAINIAN. Development-dependent Formation and Metabolism of Anthocyanins and Proanthocyanidins in Acer Species. (Under the direction of David Danehower, William Hoffmann, Jenny Xiang and De-yu Xie). Anthocyanins are one of the richest pigments, which belong to flavonoid compounds in plant kingdom. They have many biological and ecological functions. Over the past many years, numerous efforts have been made to determine the biosynthetic pathway of anthocyanins and also to identify several regulatory proteins mainly in flowers and fruits of model plants and crop plants. However, many questions concerning the metabolism of anthocyanins in foliage remains unsolved. One example is “How can developmental processes impact on accumulation patterns of anthocyanins in leaves”. In this study, we choose several cultivars from one of the most popular ornamental plants Acer palmatum Thunb. to understand the mechanism of developmental changes of pigmentation in leaf. Several other maple species were also analyzed. We propose that the metabolism of anthocyanins play an essential role in such changes. We use an integrated approach of phytochemistry and metabolic profiling to determine the biosynthesis and metabolism of anthocyanins and their impacts on foliage color. Proanthocyanidin analysis was carried out as well to determine their relationship to both anthocyanin production and foliar coloration. We have found that even for green leaves with no/trace amount of detectable anthocyanins, the biosynthetic pathway of anthocyanidin/proanthocyanidin is still activated. Our results indicate that metabolic channeling directing the anthocyanidin pathway to the proanthocyanidin biosynthesis plays a very important role in pigmentation pattern change along developmental processes. Development-dependent Formation and Metabolism of Anthocyanins and Proanthocyanidins in Acer Species by Hainian Zeng A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Plant Biology Raleigh, North Carolina 2009 APPROVED BY: _______________________________ ______________________________ Deyu Xie Jenny Xiang Committee Chair ________________________________ ______________________________ William Hoffmann David Danehower DEDICATION This thesis is dedicated to my dearest grandma, Yinchun Zhang, who passed away recently. ii BIOGRAPHY The first day I set my foot off the Airbus and onto the land of America, I knew it would be a life change. However, the thing I didn’t realize back then, is that this change has been so dramatic since that first day of air sickness and jet lag. From a metropolis to a countryside city. From Shanghai, my beloved hometown for more than 20 years, to, Raleigh, the capital city of North Carolina, one of the biggest agricultural states in the United States. This is big. Even one year later, sometimes I would still be wondering about where I was and why I was here. From time to time I was startled to see some many local people around and couldn’t help but mumbling to myself: I was in America. How time flies! Now it’s been three years, such doubt doesn’t go away but become stronger. In those three years, ups and downs are the main melody. Sometime I felt determined and I could see the bright future just ahead of me. Sometimes I felt lost and lonely, alienated myself from the outside due to serious depression. Sometimes I got sick and feared about the medical bill showing hundreds of dollars balance. Sometimes I demanded myself to be a man when facing my grandma’s past. Yes, this is life, but is it? At last, I find out what I want the most. To be with my family and friends. To fight against injustice and evil. To live a life whenever and wherever I can be myself, whenever and wherever I am in charge of my own life. At least, I won’t regret it. This is the whole point of being alive, isn’t it? Not to regret! Thanks to these three years’ life experience, I have re-founded my goal and grasped the hang of how to lead a life. To be the king of my own “land”! iii ACKNOWLEDGEMENTS I want to give my sincere appreciation and to my major advisor, Dr. Deyu Xie. He has been a wonderful mentor and advisor for these three years. Additionally, he takes care of his graduate students like his own children and treats us with respect as friends. To me, it’s not overrated to say that he is a father-like idol. The only regret I can’t let it go is that I haven’t live up to Dr. Xie’s expectations mainly due to my own weaknesses and mistakes. I’d like to give my deep gratitude to my other committee members, Drs. Bill Hoffmann, Jenny Xiang and David Danehower. They kindly gave me a lot of instructions and suggestions on my project, as well as their generous help with my life. I can still recall the time that Dr. Hoffmann gave me a ride back to my dorm, and Thanksgiving holidays I was spending in Dr. Xiang’s house as well as the knowledge of phytochemistry passed on to me by Dr. Danehower. Here I want to show my genuine gratefulness to all of my advisors for their understanding, forgiveness and noble gestures. I may be far away from being a good graduate student, but they have been continuously working with me as great advisors. And again, I’d like to apologize for my immature behaviors from time to time. I hope I can obtain your forgiveness some day. Besides my advisors, I’d like to thank our lab manager, Dr. Lili Zhou for her help with my research and her care about me. Dr. Zhou is a “mother” to us and I wish she can be with her family soon and lead a happy life ever after. Of course I would like to thank my labmates, Mingzhu, Fatima and Patrick. Without their inspiring ideas, contribution to my research and encouragements, I wouldn’t have gone this far. And last but not least, salutes to my colleagues in PBGSA for their unsinkable friendship as well. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ xi LIST OF FIGURES .................................................................................................................... xii CHAPTER 1 – LITERATURE REVIEW .................................................................................. 1 1.1 Plant flavonoids .................................................................................................................... 3 Introduction .............................................................................................................................. 3 Biosynthesis of flavonoids ........................................................................................................ 5 Ecological and physiological significances ............................................................................. 8 Nutraceutical significances .................................................................................................... 10 Taxonomic significances ........................................................................................................ 10 1.2 Anthocyanins and anthocyanidins .................................................................................... 12 Introduction ............................................................................................................................ 12 Types of anthocyanins and anthocyanidins ............................................................................ 13 Properties of anthocyanins ..................................................................................................... 15 Biosynthesis of anthocyanins .................................................................................................. 17 Ecological significances ......................................................................................................... 19 Nutraceutical significances .................................................................................................... 24 1.3 Proanthocyanidins .............................................................................................................. 25 Introduction ............................................................................................................................ 25 Types of proanthocyanidins................................................................................................ 25 Biosynthesis of proanthocyanidins ......................................................................................... 29 Ecological significances ......................................................................................................... 30 Nutraceutical significances .................................................................................................... 31 v CHAPTER 2 - FOLIAR ANTHOCYANINS AND PROANTHOCYANIDINS IN SIX ORNAMENTAL VARIETIES OF ACER PALMATUM ....................................................... 32 2.1 Introduction ........................................................................................................................ 32 2.2 Materials and methods ....................................................................................................... 39 2.2.1 Standard chemicals ............................................................................................................ 39 2.2.2 Plant materials ................................................................................................................... 39 2.2.3 Leaf collection ..................................................................................................................