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Whthesis.Pdf (1.486Mb) INVESTIGATION OF A POSSIBLE MULTI-ENZYME COMPLEX INVOLVED IN NICOTINE BIOSYNTHESIS IN ROOTS OF TOBACCO (Nicotiana tabacum) by WILLIAM HEIM Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment for the degree of MASTER OF LIFE SCIENCES in THE DEPARTMENT OF PLANT PATHOLOGY, PHYSIOLOGY, AND WEED SCIENCE with a Major Emphasis in PLANT PHYSIOLOGY APPROVED: ___________________________________ __________________ John Jelesko, Chairperson Date ___________________________________ __________________ Carole Cramer, Member Date ___________________________________ __________________ Fabricio Medina-Bolivar, Member Date August, 2003 Blacksburg, Virginia Keywords: Nicotiana tabacum, nicotine biosynthesis, diamine oxidase, N-methylputrescine oxidase, S-adenosylhomocysteine hydrolase, multi-enzyme complex, metabolon 2003 by William Heim ALL RIGHTS RESERVED INVESTIGATION OF A POSSIBLE MULTI-ENZYME COMPLEX INVOLVED IN NICOTINE BIOSYNTHESIS IN ROOTS OF TOBACCO (Nicotiana tabacum) by William Heim (Dr. John Jelesko, Chairperson) Department of Plant Pathology, Physiology, and Weed Science ABSTRACT N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a “channel” without equilibrating with the bulk solution (Ovádi, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs. ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. John Jelesko for his indispensable guidance during the course of my research and writing of this thesis. Drs. Carole Cramer and Fabricio Medina-Bolivar provided excellent and timely advice during committee meetings as well as informal hallway and lab discussions. As I found out during my final oral defense, they were also intent on making sure that I passed the test the old-fashioned way, i.e., I had to EARRRN it. Deborah Reed deserves thanks for orienting me in the Jelesko Lab when I arrived in July ’01, and for providing help, advice, and great primers later during my research. Laboratory research assistant Elizabeth Tucker demonstrated how to do Northern blots, and as a result of her good example I was able to produce the high quality results that appear in Chapters 3 and 4. Undergraduate research assistant Amanda Melillo put in a lot of time growing up, harvesting, and freezing the tobacco root tissue used for some of the Northern blot analyses. Mr. Chengsong Zhao of the Beers Lab in the Horticulture Department at Virginia Tech provided the single most important piece of practical assistance when he guided the design of the degenerate primers that were finally successful (after several unsuccessful attempts using primers of our own design) in identifying a fragment of a gene which we hope will prove to be N-methylputrescine oxidase (MPO) when all is said and done. Finally, I thank God, who patiently carried me through the difficult times that arose during the course of my research, and who gave me the privilege of participating in the revealing of what may prove to be new insights into His handiwork in nature, as detailed in this thesis. iii TABLE OF CONTENTS ABSTRACT .......................................................................................................................................ii ACKNOWLEDGMENTS ................................................................................................................ iii TABLE OF CONTENTS ................................................................................................................iv LIST OF FIGURES .........................................................................................................................vi LIST OF TABLES............................................................................................................................vii LIST OF ABBREVIATIONS AND ACRONYMS ....................................................................... viii CHAPTER I Literature Review ....................................................................................................1 I.1 LITERATURE REVIEW ......................................................................................................2 LITERATURE CITED......................................................................................................................5 CHAPTER II Cloning of SAHH from Tobacco cDNA Library.................................................7 II.1 ABSTRACT ..........................................................................................................................8 II.2 INTRODUCTION .................................................................................................................8 II.3 MATERIALS AND METHODS ........................................................................................9 II.3.1 Collection of Seed Stock...............................................................................................9 II.3.2 Preparation of Root Cultures.......................................................................................10 II.3.3 Purification of Polyclonal ‘anti-MPO’ Antibody........................................................10 II.3.4 Screening of Tobacco Root cDNA Expression Library for MPO ..............................11 II.3.5 Nucleotide Sequencing of Candidate MPO DNA Inserts ...........................................12 II.4 RESULTS AND DISCUSSION..........................................................................................13 II.4.1 Screening of Tobacco Root cDNA Expression Library for MPO and Nucleotide Sequencing of Candidate MPO DNA Inserts..............................................................13 LITERATURE CITED....................................................................................................................26 CHAPTER III Investigation of a Possible Multi-enzyme Complex Involved in Nicotine Biosynthesis in Roots of Tobacco ........................................................................27 III.1 ABSTRACT ........................................................................................................................28 III.2 INTRODUCTION ...............................................................................................................28 III.3 MATERIALS AND METHODS ......................................................................................30 III.3.1 Collection of Seed Stock.............................................................................................30 III.3.2 Preparation of Root Cultures.......................................................................................30 III.3.3 DAO Enzyme Assay and Immunodepletion...............................................................30 III.3.4 RNA Extraction and Northern Blot Analysis..............................................................33 III.3.5 Protein Extraction and Immunoblot Analysis.............................................................35 III.4 RESULTS............................................................................................................................36 III.4.1 DAO Enzyme Assay and Immunodepletion...............................................................36 III.4.2 Steady State mRNA Levels for Selected Genes in Nicotine Biosynthesis and SAM Recycling Pathways....................................................................................................42 III.4.3 Protein Extraction and Immunoblot Analysis.............................................................46
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