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Enhancement of the Free Amino Acid and Protein Content Of ENHANCEMENT OF THE FREE AMINO ACID AND PROTEIN CONTENT OF CASSAVA STORAGE ROOTS AND EVALUATION OF ROOT-SPECIFIC PROMOTERS IN CASSAVA. DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Elisa Leyva-Guerrero Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2011 Dissertation Committee: Dr. Patrice Hamel, Advisor Dr. James Metzger Dr. Richard Sayre, Co-Advisor Dr. Randy Scholl Dr. Dave Somers Copyright by Elisa Leyva-Guerrero 2011 ABSTRACT Cassava is an important staple crop for millions of people around the world in particular in Sub-Saharan Africa. Cassava is preferred given its many agronomical attributes such as ability to grow in poor soils and drought resistance. Cassava storage roots are also a good source of calories (starch), however, they are deficient in protein and other micronutrients such as iron and zinc. Protein malnutrition is widespread in the regions were cassava is widely consumed and consumption of cassava as a staple has been linked to reduced protein intake in the diet. A cassava storage root with higher protein could potentially impact the nutrition and well being of millions people. There are several proposed strategies to increase the protein and free amino acid content in crops such as modification of the amino acid biosynthetic pathways and over expression of native storage proteins. In this thesis we propose two different approaches for increasing free amino acids and protein. One approach was to increase nitrate assimilation through the expression of a mutated nitrate reductase. Nitrate reduction to nitrite is followed by a further reduction to ammonium which can be readily incorporated into amino acids. A rate limiting step in nitrate metabolism is the reduction of nitrate to nitrite by nitrate reductase. This enzyme is highly regulated, however, it has been observed that through mutation of a key regulatory serine the enzyme remains active and the assimilation of nitrate increases resulting in increased free amino acid levels. In ii cassava the root-specific expression of a mutated nitrate reductase resulted in a the doubling of the storage root free amino acid content, however, no root protein increase was observed. A second approach was to utilize the nitrile group present in the cassava cyanogenic glucoside linamarin as a reduced nitrogen source. The hydrolysis of linamarin releases acetone cyanohydrin which in turn can degrade to release cyanide. In all plants there is a cyanide assimilation pathway involving β-cyanoalanine synthase, the end products of this pathway are aspartate and ammonia. Through increased hydrolysis of linamarin, we sought to increase the assimilation of cyanide in to aspartate and ammonia. Both of these compounds can be utilized for the synthesis of additional amino acids. The linamarase enzyme is responsible for linamarin hydrolysis. In wild-type cassava the interaction between linamarase and linamarin is limited spatially; linamarase is found in the cell wall and linamarin in the vacuole. Through the expression of a vacuolar targeted linamarase, we proposed to increase the hydrolysis of linamarin and as a result provide more reduced nitrogen (nitrile) for free amino acid and protein synthesis. The expression of a vacuolar linamarase in cassava storage roots resulted in a doubling of the free amino acid pool in this organ but no increase in protein. It was only through the dual expression of vacuolar linamarase and the storage protein sporazein that both an increase in free amino acid and protein in the storage roots was observed. Interestingly the expression of a mutated nitrate reductase and the expression of a vacuolar linamarase with or without sporazein in the cassava storage roots resulted in an increase in free amino acid and protein levels in the leaves. This indicates the presence of amino acid transport from the iii storage roots to a strong nitrogen utilization organ in this case the leaves where the amino acids are incorporated in to protein. Importantly even with elevated root expression of sporazein the leaves had increased free amino acids and protein indicating that the expression of a root storage protein is not sufficient to substantially reduce leaf nitrogen sink strength. Although cassava is a main staple food the molecular biology tools available for its transformation are very limited. In the final chapter of this thesis we evaluated four different Arabidopsis root promoters in cassava to determine their functionality and tissue specificity. Two promoters named A14 and E40 were determined to be functional in cassava roots with minimal leaf expression. The availability of an increased set of root promoters for cassava may increase the development of transgenic cassava with increased nutritional and industrial value. iv DEDICATION Too the most wonderful and dedicated parents in the world v ACKNOWLEDGEMENTS I thank first and beyond anyone God, for giving me life, for giving me a wonderful family and friends and for being a constant presence and a source of support. I thank my two wonderful and dedicated parents Roberto and Rosa Maria for it is because of them I have made it this far. I am a regular person who had great parents believing in her, believing that I could make it this far. Thank you for everything. I thank my advisor Dr. Richard Sayre for taking me in his lab, for helping me grow and being there throughout the years. Thank you for allowing me to pursue this dream and for all your guidance. I thank my little sister Alejandra for always being a loving sister and someone who helped me keep my spirits high at all times. I thank my very soon to be husband Andy for all his love in this last years and specially in these last months, for taking care of little things and big things and making a big difference in my life. vi I thank all my grandmothers Pita and Virginia and my grandfathers Roberto and Jacobo for their presence in life or from heaven, thank you for your love. I thank all my family, my aunts and uncles, all my cousins, thank you for always believing in me. I thank Adriana for her friendship throughout all these years, for your kindness and for always being there for me. I thank Luis for his friendship in grad school and beyond, for always listening to me no matter what time of day or length of phone call, for making me laugh whether it was intentional or not. I thank Yuriko for being my surrogate little sister when our own sisters were away, thank you for your friendship. I thank my labmates that are more than that, my friends Vanessa, Zoee, Anil and Narayanan, I don’t know how I could have made it without you guys, thank you in particular to Narayanan for all his guidance and help with my projects and Vanessa for reading my thesis at the last minute. I thank all those that have gone through the Sayre Lab, those that have taught me and those that have allowed me to teach, in particular to Uzo for teaching me cassava subculturing. I would like to thank also Dr. Nigel Taylor for teaching me cassava techniques. I thank the Keppler family for making me feel so welcomed and for all the family outings that have made these last years so much more enjoyable. To all my friends a long long vii list to go through, but I thank my friends in Mexico for their long distance friendship and your encouragement as always on facebook or by email, you don’t know how much it meant to me. I thank Ro for always caring, for being there no matter what, for letting me vent and listening. To Niro for being my first friend in Columbus and for always being so kind. I thank the Jose’s and Sergio for the dancing and partying nights that made life in Columbus all the much livelier, Roxana for being my super concert friend and for letting me stay with her all the time in Columbus and because no matter what we had to do we always managed to sneak to Chicago to see Julieta. To Shely and her family, the Zakins and the Fans for opening your homes to me. To Nadja and Xuan, for being my first friends in St. Louis. To all my friends in St. Louis and all over the world! I thank also all my friends in dachshund and dog rescue land. viii VITA 1981…………Born- San Luis Potosi, SLP, Mexico 2004………….B.S. Chemistry, Universidad Autonoma de San Luis Potosi 2004-2011……Graduate Research Associate, The Ohio State University FIELD OF STUDY Major Field: Plant Cellular and Molecular Biology ix TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION .................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................... vi VITA .................................................................................................................................. ix FIELD OF STUDY ............................................................................................................ ix LIST OF FIGURES……………………………………………………………………..xix LIST OF TABLES…………………………………………………………………..…xxiv CHAPTER 1 INTRODUCTION ........................................................................................ 1 1.1 BIOLOGY OF CASSAVA ....................................................................................... 1 1.2 CASSAVA IN AFRICA ..........................................................................................
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