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Elucidation of late steps in pisatin biosynthesis Item Type text; Dissertation-Reproduction (electronic) Authors DiCenzo, Gregory Lawrence Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 06/10/2021 04:13:37 Link to Item http://hdl.handle.net/10150/282830 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 ELUCIDATION OF LATE STEPS IN PISATIN BIOSYNTHESIS by Gregory Lawrence DiCenzo A Dissertation Submitted to the Faculty of the DEPARTMENT OF PLANT PATHOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 199 8 UMI Number: 991213 7 UMI Microform 9912137 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 2 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Gregory Lawrence DlCenzo entitled Elucidation of Late Steps in Pisatin Biosynthesis and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Date Date Date Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requitfeii Dissertation Director 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial ftilfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED 4 ACKNOWLEDGMENTS I especially acknowledge and thank Hans VanEtten for his generous support, help and confidence. I also thank Merritt Nelson and the Department of Plant Pathology for their support, particularly in the late stages. Two individuals stand out from the others with whom Tve worked over the years and they deserve special thanks and acknowledgment They are Lajos Szabo, formerly of the University of Arizona Chemistry Department, and Qindong Wu, longtime friend and, while I was working in the lab, molecular techniques teacher. It is fitting that they have both gone on to bigger and better things. 5 Dedicadon This dissertation has been dedicated all along to my son Michael DiCenzo. Today is his birthday: he turns ten. Michael, years ago, when I knew this Ph.D. would take me no where, I stuck it out in order to back up a future plea by me, to you, to not give up on something. I promise not to use this device except in the most important and desperate of times. Thank you for being such a wonderful young man. Your father November 14, 1998 6 TABLE OF CONTENTS LIST OF FIGURES 8 LISTOFTABLES 10 ABSTRACT 11 CHAPTER I: INTRODUCTION 13 ROLE FOR PHYTOALEXINS 14 ISOFLAVONOIDS AND PISATIN BIOSYNTHESIS 20 LATE STEPS OF PISATIN BIOSYNTHESIS 32 SUMMATION OF (+) PISATIN BIOSYNTHESIS 39 CONCLUSION 40 CHAPTER 2: In vivo Incorporation of Isoflavonoids into Pisatin in Pea 46 ABSTRACT 46 INTRODUCTION 48 MATERIALS AND METHODS 56 Plant material 56 Chemicals 56 Induction of pisatin biosynthesis and substrate administration 58 Analysis of label incorporation 58 Metabolite analysis 59 Thin layer chromatography 59 High-performance liquid chromatography 59 Quantification of radiolabel incorporation 60 RESULTS 60 Time course of pisatin production in pea cotyledons treated with CuCl2 61 Incorporation of tritiated (+) sophorol and (+) maackiain 61 Isoflavone as precursor 66 Incorporation of (-) sophorol 69 Relative efficiency of incorporation 69 Metabolism of DMDI 75 Identity of the pisatin product 80 DISCUSSION 81 CHAPTER 3: ISOLATION OF PISATIN BIOSYNTHETIC INTERMEDIATES FROM PEA. 89 ABSTRACT 89 INTRODUCTION 90 MATERIALS AND METHODS 96 Plant materials 96 Chemicals 96 Enzyme assays 96 Analytical methods 97 NMR, Mass Spectroscopy 98 In vivo utilization of P18 98 7 TABLE OF CONTENTS - Continued RESULTS 100 Reaction products of (±) sophorol produced by protein extracts of CuCl2-inciuced pea seedlings 100 Metabolism of (-) sophorol by pea protein 100 MS analysis of the sophorol metabolites: P8 110 MS analysis of the sophorol metabolites: P18 110 IHNMR ofP18 (500MHz) 121 HMBC and Inverse mode HMQC 121 Analysis of 1 la-6a configuration by circular dichroism 132 /n VIVO utilization of P18 137 /nvifro utilization of P18 137 DISCUSSION 142 Identification ofP8 as isoflavene and P18 as rronj-HMK 142 Formation of DMDI in vitro from (-) sophorol 145 Metabolism of (+) sophorol by pea 148 Metabolism of franj-HMK by pea 149 Role of rra/ty-HMK in pisatin biosynthesis 149 CHAPTER 4: ISOLATION OF A CDNA ENCODING (-) SOPHOROL REDUCTASE WHICH IS INVOLVED IN PHYTOALEXIN BIOSYNTHESIS IN PEA 153 ABSTRACT 153 INTRODUCTION 155 MATERIALS AND METHODS 161 Plant materials and chemicals 161 Screening of the cDNA library 161 Sequencing of sophorol reductase cDNA fragments 162 PGR amplication and cloning of sophorol reductase cDNA 162 Northern analysis 163 Genomic DNA characterization and Southern analysis 164 Expression of isoflavanone reductase (SOR) activity in E. coli 166 RESULTS 167 Isolation of SOR clones 167 Northern analysis 167 Genomic analysis 174 Expression of sophorol reductase in E. coli 177 DISCUSSION 184 APPENDIX A 188 REFERENCES CITED 190 8 LIST OF FIGURES Figure 1.1: (+) Pisatin 15 Figure 1.2; Flavonoid biosynthesis 21 Figure 1.3: Isoflavone biosynthesis 24 Figure 1.4: Mechanism of reduction by isoflavone reductase 27 Figure 1.5: Biosynthesis of isoflavonoids in alfalfa 30 Figure 1.6: Model of (+) pisatin biosynthesis after Dewick 34 Figure 1.7: 6a-hydroxylation of pterocarpans in pea and soybean 36 Figure 1.8: Summary of results presented in this dissertation 42 Figure 2.1: Model of (+) pisatin and (-) maackiain biosynthesis after Banks and Dewick 49 Figure 2.2: Empirical features of (+) pisaun and medicarpin biosynthesis 52 Figure 2.3: Production of pisatin by wounded pea cotyledons 62 Figure 2.4: In vivo incorporation of (+) maackiain and (+) sophorol into pisatin 64 Figure 2.5: In vivo incorporation of DMI, (-) sophorol, (+) sophorol and (-) maackiain into pisatin 67 Figure 2.6: In vivo incorporation of (-) sophorol into pisatin 70 Figure 2.7: Efficiency of incorporation of (+) and (-) sophorol 73 Figure 2.8: Metabolism of DMDI 76 Figure 2.9: Chirality of tritiated pisatin 78 Figure 2.10: Acid-treated pH] pisatin 82 Figure 3.1: Model of the (+) pisatin biosynthetic pathway based on in vivo labeling 91 Figure 3.2: Late steps in phytodexin biosynthesis of pea, alfalfa and soybean 93 Figure 3.3: //z v/rro metabolism of (±) pH] sophorol 101 Figure 3.4: In vitro metabolism of (-) and (+) sophorol 103 Figure 3.5: Metabolism of (-) sophorol (HPLC) 106 Figure 3.6: Metabolism of (-) sophorol (TLC) 108 Figure 3.7: /n virro metabolism of DMDI Ill Figure 3.8: Absorbance scan of P8 113 Figure 3.9: Mass spectra of P8 and authentic isoflav-3-ene 115 Figure 3.10: EI Mass spectra of P18 117 Figure 3.11: UV absorbance of P18 and cw-HMK 119 Figure 3.12: CI+MSofP18 122 Figure 3.13: 500MHz IH-NMR spectrum of P18 125 Figure 3.14: 250MHz IH-NMR spectrum of m-HMK 128 Figure 3.15: Hydrogen carbon connections

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