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The Nonprotein Amino Acid Meta-Tyrosine: Its Biosynthesis, Phytotoxicity, and Application As a Tool for Research on Aromatic Amino Acid Metabolism in Plants

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The Nonprotein Amino Acid Meta-Tyrosine: Its Biosynthesis, Phytotoxicity, and Application As a Tool for Research on Aromatic Amino Acid Metabolism in Plants The nonprotein amino acid meta-tyrosine: Its biosynthesis, phytotoxicity, and application as a tool for research on aromatic amino acid metabolism in plants by Tengfang Huang This thesis/dissertation document has been electronically approved by the following individuals: Jander,Georg (Chairperson) Hua,Jian (Minor Member) Owens,Thomas G (Minor Member) THE NONPROTEIN AMINO ACID META-TYROSINE: ITS BIOSYNTHESIS, PHYTOTOXICITY, AND APPLICATION AS A TOOL FOR RESEARCH ON AROMATIC AMINO ACID METABOLISM IN PLANTS A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Tengfang Huang August 2010 © 2010 Tengfang Huang THE NONPROTEIN AMINO ACID META-TYROSINE: ITS BIOSYNTHESIS, PHYTOTOXICITY, AND APPLICATION AS A TOOL FOR RESEARCH ON AROMATIC AMINO ACID METABOLISM IN PLANTS Tengfang Huang, Ph. D. Cornell University 2010 In addition to the twenty amino acids that are universally found as building blocks of proteins, nonprotein amino acids are also present in numerous plant species. One such nonprotein amino acid, meta-tyrosine, is abundant in the root exudates of Chewings fescue (Festuca rubra L. ssp. commutata). Consistent with the phytotoxic effects of m- tyrosine, prior studies showed that Chewings fescue has superior weed-suppressive capacity in field trials involving dozens of turf grass varieties. Further research with m-tyrosine demonstrates that this compound is stored in a different compartment than the protein amino acids, and can be easily washed off fescue roots with aqueous solutions. Chewings fescue roots have enzymatic activity, likely from a cytochrome P450, for synthesizing m-tyrosine from phenylalanine. An EST library was constructed from roots of Chewings fescue and four candidate cytochrome P450 genes are identified and cloned. When added to A. thaliana growth medium, m-tyrosine can be misincorporated into A. thaliana proteins. Several protein amino acids, in particular phenylalanine, partially rescue the toxicity of m-tyrosine. Correspondingly, a screen for m-tyrosine resistance in A. thaliana identified a mutant that over-accumulates free phenylalanine. Map-based cloning showed that the genetic basis of this phenotype is adt2-1D, a mutant allele of ADT2, which encodes arogenate dehydratase, the final enzyme of the phenylalanine biosynthesis pathway. Characterization of ADT2 and its mutant form revealed feed-back regulation of phenylalanine biosynthesis, which depends on a critical motif of the ADT2 protein. Overexpression of the feedback-insensitive adt2- 1D produces even higher levels of free phenylalanine and has pleiotropic physiological consequences, including abnormal leaf development, resistance to 5- methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper), and increased salt tolerance. Finally, several hypotheses for m-tyrosine modes of action were tested and discussed. BIOGRAPHICAL SKETCH Tengfang Huang grew up in Fujian Province on China’s southeast coast. After finishing study at Guoguang High School, he went to Fudan Univsersity in Shanghai, China, where he received a Bachelor’s degree in Biological Sciences in 2004. During his senior year at college, he participated in an academic exchange program at Hong Kong University of Science and Technology, and started the research on Arabidopsis thaliana. In 2004, Tengfang Huang was admitted into the Ph.D. program at Department of Plant Biology, Cornell University. After a year of rotation, he joined Dr. Georg Jander’s lab at Boyce Thompson Institute in 2005 and focused his research on plant amino acid metabolism. He was married to Ms. Jing Zhou, a fellow Cornell graduate student. iii 献给我的家人，特别是我的外祖父黄英标先生 (1921-2009) Dedicated to my family, especially my grandpa Mr. Yingbiao Huang (1921-2009) iv ACKNOWLEDGMENTS I thank my advisor Georg Jander for his continuous support, inspiration and encouragement. I would also like to extend my sincere thanks to my other committee members, Tom Owens and Jian Hua, for their guidance and advice. I thank many lab members in the Jander lab, especially Martin de Vos, for his discussion and suggestions. I thank Lythia Hoffstaetter for working with me over two summers, and Ludmila Rehak for her pioneering work on m-tyrosine biosynthesis. I am grateful to Alistair Fernie and two of his lab members, Takayuki Tohge and Anna Lytovchenko, for hosting my visit to Max-Planck-Institute for Molecular Plant Physiology and assistance with the metabolite profiling. I also want to express my thanks to Li Foundation, who generously awarded me a Li Foundation Fellowship during my first year at Cornell, and other funding agencies including National Science Foundation, Triad Foundation, and BARD (Binational Agricultural Research and Development Fund) for providing grants to our research. Finally, I want to give special thanks to my parents Jialiang Huang and Shumian Huang, and my wife Jing Zhou, for their love and endless support. v TABLE OF CONTENTS BIOGRAPHICAL SKETCH ......................................................................................... iii DEDICATION .............................................................................................................. iv ACKNOWLEDGMENTS .............................................................................................. v LIST OF FIGURES ....................................................................................................... ix LIST OF TABLES ........................................................................................................ ix CHAPTER ONE: OVERVIEW OF NONPROTEIN AMINO ACIDS IN PLANTS .... 1 Occurrence and biosynthesis of nonprotein amino acids ........................................... 1 Functions of nonprotein amino acids in plant defense to insect attacks ..................... 2 Canavanine ............................................................................................................. 2 γ - Aminobutyric Acid (GABA) ............................................................................. 5 β-Aminobutyric Acid (BABA) ............................................................................... 6 Other Nonprotein Amino Acids and Their Common Themes ............................... 7 Studies of amino acid metabolism using nonprotein amino acids ............................. 9 meta-Tyrosine and this thesis ................................................................................... 10 CHAPTER TWO: META-TYROSINE IN FESTUCA RUBRA SSP. COMMUTATA (CHEWINGS FESCUE) IS SYNTHESIZED BY HYDROXYLATION OF PHENYLALANINE ..................................................................................................... 19 Introduction .............................................................................................................. 19 Results and discussion .............................................................................................. 21 Distribution of m-tyrosine in F. rubra roots ......................................................... 21 Phenylalanine is a direct precursor for m-tyrosine biosynthesis .......................... 23 Conversion of phenylalanine to m-tyrosine is independent of fescue endophytes and is highest in the roots ..................................................................................... 25 Conversion of phenylalanine to m-tyrosine is reduced by cytochrome P450 inhibitors ............................................................................................................... 27 Identification of F. rubra cytrochrome P450 genes ............................................. 27 Concluding remarks .................................................................................................. 30 Experimental ............................................................................................................. 30 Material and growth conditions ............................................................................ 30 Analysis of free amino acids by HPLC ................................................................ 31 vi Analysis of isotope-labeled free amino acids by GC-MS .................................... 31 Fescue root cDNA library construction ................................................................ 32 Sequence analysis ................................................................................................. 32 RT-PCR ................................................................................................................ 33 Cloning and expression of candidate cytochrome P450 cDNAs .......................... 33 References ................................................................................................................ 35 CHAPTER THREE: PLEIOTROPIC PHYSIOLOGICAL CONSEQUENCES OF FEEDBACK-INSENSITIVE PHENYLALANINE BIOSYNTHESIS IN ARABIDOP SIS THALIANA* ........................................................................................ 38 Summary ................................................................................................................... 38 Introduction .............................................................................................................. 39 Results ...................................................................................................................... 42 Selection for A. thaliana mutants resistant to m-tyrosine ..................................... 42 Identification of a point
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