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Proquest Dissertations Expression and regulation of phytoene desaturase during maize seed development Item Type text; Dissertation-Reproduction (electronic) Authors Hable, Whitney Elizabeth, 1967- 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 09:37:56 Link to Item http://hdl.handle.net/10150/282172 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UME films the text directly fi-om 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. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Kgher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zedj Road, Ami Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 EXPRESSION AND REGULATION OF PHYTOENE DESATURASE DURING MAIZE SEED DEVELOPMENT by Whitney Elizabeth Hable A Dissertation Submitted to the Faculty of the GRADUATE INTERDISCIPLINARY PROGRAM IN GENETICS In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1996 DMI Niomber: 9713407 UMI Microform 9713407 Copyright 1997, 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 Whitney Elizabeth Hable entitled Expression and Regulation of Phvtoene Desaturase Dririnq Maize Seed Development and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Datf/ ^ / Date Date C/MIL ite 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 requirement. Dissertation Dir« or 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment 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: 0 4 ACKNOWLEDGMENTS There are a number of people I wovild like to thank for their encouragement and friendship during my time at The University of Arizona. I especially thaiik my advisor, Karen Oishi, for her wonderful abilities as a mentor and a scientist, and her remarkable patience, giving her students room to leam and grow on their own. Karen has influenced my development tremendously both as a scientist and as an individual, stressing the importance of critical thinking and of sharing knowledge with others through writing, presentations, and teaching. These fundamental skills are important to me and I hope that I will continue to leam from her. I would also like to thank the other members of my committee, Carol Dieckmann, Brian Larkins, Karen Schumaker, and Hai\s van Etten for their support during my graduate studies. This diverse committee was invaluable for helping me think about my project more critically and from new perspectives. I am equally indebted to them for their support as I finished my PhD with a long-distance advisor. I want to thank Karen Schumaker specifically for helping Karen Oishi advise me while she was away. Karen Schumaker helped to keep me focused on finishing and planning my future. Through attending Karen's lab meetings I also enjoyed the opportunity to leam different scientific approaches upon which her research is based, and which have influenced my postdoctoral decision. I am extremely greatful for Karen's support and her willingness to spend her time helping me figure out what's next. Additionally, I thank the graduate students and postdocs who are my peers and friends, for their support. My lab-mates, Kathy Moore, Bob Sheehy, A1 Agellon and Carol Mahaffey were invaluable for their help and advice in all aspects of graduate school. I also enjoyed the friendship and support of Clare Beelman, Jermifer Swiergiel, Margaret Dietrich, Ken Helm, Kristin Jenkins, Mike Jerxkins, Teri Suzuki and Nadi Weymeyer. Giordi, tharik you for your patience, love and support. Your continuing confidence in my abilities, scientific and otherwise, has been on of the most important elements of my progress in these last five years. None of this could be said or done without the encouragement of my parents, grandparents and sisters who instilled in me the importance of education and of doing what makes me happy. Thank you all! 5 TABLE OF CONTENTS LIST OF ILLUSTRATIONS 7 ABSTRACT 10 CHAPTER L LITERATURE REVIEW AND INTRODUCTION 12 Developmental importance of regulated metabolites 13 Significance of the metabolite abscisic add 14 Role of biosjmthesis in ABA accumulation 18 Carotenoid biosynthesis 22 Phytoene desaturase 25 Hypothesis 29 Maize seed development 30 Regulation of the PDS transcript and protein 34 Roles of ABA biosynthetic mutants 39 CHAPTER II. CLONING AND CHARACTERIZATION OF MAIZE PDS 42 Introduction 43 Results 48 Discussion 62 CHAPTER III. PDS TRANSCRIPT AND PROTEIN LEVELS DURING SEED DEVELOPMENT 64 Introduction 65 Results 68 PDS transcript levels 68 PDS protein levels 76 Discussion 88 CHAPTER IV. THE VPS LOCUS ENCODES PDS 93 Introduction 94 Results 97 Genetic mapping 97 Pds alleles in the vp5 mutant 98 PDS transcript in the vp5 mutant 110 PDS protein in the vp5 mutant 113 Discussion 117 6 TABLE OF CONTENTS-Continued CHAPTER V. THE ROLES OF SOME VIVIPAROUS MUTANTS IN ABA BIOSYNTHESIS 119 Introduction 120 Results 122 Role of vp2 122 Role of lycopene 125 Discussion 133 CHAPTER VI. GENERAL DISCUSSION 136 Goals and rationale 137 Models for regulation of ABA accumulation 137 vp5 encodes PDS 144 Roles of VP2 c:nd lycopene 144 Summary 145 CHAPTER VII. MATERIALS AND METHODS 146 Plant material 147 Nucleic acid preparation 148 RNA isolation 148 Genomic DNA isolation 150 Plasmid DNA isolation 151 Amplification, cloning and sequencing of maize and soybean PDS 151 cDNA library screening and sequence analysis 152 RNA hybridization analysis 153 Genomic DNA hybridization analysis 154 PDS fusion protein generation and pvirification 156 Chicken polyclonal antibodies to Pt3S 158 Rabbit polyclonal antibodies to PDS 159 Total and plastid membrane-enriched protein isolation 160 Immimoblot analysis 160 Affinity purification of antibodies to the PDS fusion protein 161 REFERENCES 162 7 LIST OF ILLUSTRATIONS Figure 1.1, Direct and indirect pathways of ABA biosynthesis in higher plants 20 Figure 1.2, The indirect pathway of carotenoid and ABA biosynthesis in higher plants 26 Figure 1.3, Maize seed development 31 Figtire 1.4, ABA levels in maize seeds 35 Figure 2.1, Alignment of the predicted PDS protein sequences of tomato and soybean 46 Figure 2.2, Nucleotide sequence of the full-length maize PDS cDNA 49 Figure 2.3, Comparison of the predicted PDS proteins from maize and pepper 53 Figure 2.4, Comparison of the predicted PDS proteins from higher plants, cyanobacteria, bacteria, and fungi 55 Figure 2.5, PDS transcript levels in maize tissues 60 Figure 3.1, PDS transcript levels in maize endosperm and embryos grown in 1991 ' 70 Figure 3.2, Normalized PDS transcript levels in maize endosperm grown in 1991 and 1993 72 Figure 3.3, Normalized PDS transcript levels in maize embryos grown in 1991 and 1993 74 Figure 3.4, Normalized PDS transcript levels in maize embryos and endosperm 77 Figure 3.5, Immimoblot analysis of PDS from maize leaves 80 8 LIST OF ILLUSTRATIONS-Conftnued Figure 3.6, Immimoblot analysis of PDS from maize leaf, embryo
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