University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2008 Understanding polyamine metabolism through transgenic manipulation in poplar suspension cultures Sridev Mohapatra University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Mohapatra, Sridev, "Understanding polyamine metabolism through transgenic manipulation in poplar suspension cultures" (2008). Doctoral Dissertations. 431. https://scholars.unh.edu/dissertation/431 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. UNDERSTANDING POLYAMINE METABOLISM THROUGH TRANSGENIC MANIPULATION IN POPLAR SUSPENSION CULTURES By SRTOEV MOHAPATRA B.S. Sambalpur University, Sambalpur, India, 1999 M.S. Utkal University, Bhubaneswar, India, 2001 DISSERTATION Submitted To the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Plant Biology May, 2008 UMI Number: 3308381 INFORMATION TO USERS 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 bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send 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. UMI UMI Microform 3308381 Copyright 2008 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 E. Eisenhower Parkway PO Box 1346 Ann Arbor, Ml 48106-1346 This dissertation has been examined and approved, V Dissertation Director, Subhash C. Minocha Professor of Plant Biology and Genetics Rakesh Minocha Research Biochemist, USDA-Forest Service Affiliate Professor of Plant Biology and Natural Resources, UNH Curtis V. Givan Professor of Plant Biology Leland h Professor of Plant Biology Andrew Laudano Associate Professor of Biochemistry and Molecular Biology Date Pl^kof &"b$- DEDICATION This dissertation is dedicated to my family: my grandfather, my parents, my brother, my sister-in-law, my wife and my little niece. 111 ACKNOWLEDGEMENTS My sincere gratitude goes to my dissertation director, Dr. Subhash C. Minocha and my (almost) dissertation co-director, Dr. Rakesh Minocha for giving me this opportunity to conduct this research and for their constant guidance, support and patience through my entire journey as a Ph.D. student. Thanks to Dr. Ed Herbst for initiating research on polyamines at UNH which was then carried forward by Drs. Rakesh Minocha and Subhash Minocha. I also thank with all my heart, the past and present members of Dr. Rakesh Minocha's lab at the USDA-Forest Service, namely, Stephanie Long, Dr. Palaniswamy Thangavel and Benjamin Mayer for providing me with technical support and carrying out several of the analyses used in this research. Thanks to the other members of my dissertation committee, Dr. Leland Jahnke, Dr. Curtis V. Givan and Dr. Andrew P. Laudano for their guidance and feedback. Thanks to the past and present members of the Minocha lab at UNH, namely, Dr. Pratiksha Bhatnagar, Dr. Andrew Page, Dr. Sridevi Ganapathi, Jeff Mitchell, Rob Chretien, Brenna Parsons, Kathryn Tiberghein, Michelle Serapiglia, Challa Vasavi, Chaz "the dude" Rice, Smita Singh and Rajtilak Majumdar for all their help over the years. Thanks to Kathryn Chervinky and Dr. Arnab Sen for their help with troubleshooting some of the molecular techniques and to Priya Wiley for her help with some of the enzyme assays and statistical analyses. Thanks to Dr. Leland Jahnke and Dr. Ed Tillingharst for letting me use some of the chemicals from their labs. Thanks to Dr. Carl Vaughan for his constant support and encouragement while I served as TA for biology labs. Thanks to the Dept. of Plant Biology for partially iv funding this research and for providing such a good support system. Thanks to all the graduate students and faculty members in Rudman Hall for their help from time to time. Thanks to my family and all my friends who have stuck with me through thick and thin. Last but not the least, I want to thank my wife Aiswarya for all the love, support and encouragement she has pampered me with and for putting up with me all these years. This research was co-funded by USDA-NRI, USDA-Forest Service and Dept. of Plant Biology, UNH. v TABLE OF CONTENTS DEDICATION... iii ACKNOWLEDGEMENTS iv LIST OF TABLES xi LIST OF FIGURES xii ABBREVIATIONS xvi ABSTRACT xviii GENERAL INTRODUCTION 1 What are polyamines? 1 Why study polyamine metabolism? 2 Metabolism of polyamines and related amino acids 5 Genetics of polyamine metabolism in plants 7 Genetic manipulation of polyamine metabolism 9 Functions of polyamines 17 Polyamines in growth and development 17 Polyamines and stress 20 Polyamines as indicators of stress in trees 22 Current status of polyamine research in our laboratory 24 Objectives of the present study 25 Suspension culture vs. intact tissues 26 GENERAL MATERIALS AND METHODS 28 Cell culture and harvest 28 Free polyamine analysis 29 Amino acid analysis 30 Determination of reduced glutathione (GSH), the phytochelatin PC2 and y- glutamylcysteine (y-EC) 31 Ion analysis : 32 Measurement of mitochondrial activity and membrane integrity 33 VI Measurement of total protein content 33 Genomic DNA isolation and quantification 34 Polymerase Chain Reaction (PCR) 35 Statistical Analysis 36 CHAPTER 1 37 PUTRESCINE OVERPRODUCTION AND ACTIVITIES OF THE POLYAMINE BIOSYNTHETIC ENZYMES 37 Introduction 37 Materials and Methods 39 ODC, ADC and SAMDC enzyme assays 39 Results 41 Confirmation of stable transformation 41 Cellular contents of polyamines over the 7 d culture cycle 41 Cellular contents of soluble proteins over the 7 d culture cycle 44 Activities of ODC, ADC and SAMDC over the 7 d culture cycle 45 Expression of different genes over the 7 d culture cycle 48 Discussion . 48 Conclusions 58 CHAPTER II 59 ENHANCED PUTRESCINE METABOLISM AND SOLUBLE AMINO ACIDS.. 59 Introduction 59 Materials and Methods 63 Total organic carbon and nitrogen 63 Results 63 Amino acids derived from a-ketoglutarate (the Glutamate family) 63 Amino acids derived from 3-phosphoglycerate (3-PGA) 66 The aromatic amino acids (derived from phosphoeno/pyruvate) 66 Amino acids derived from pyruvate (the pyruvate family) 68 Amino acids derived from oxaloacetate (the aspartate family) 70 Gamma-aminobutyric acid (GABA) 73 Total carbon and nitrogen 73 Discussion 73 Conclusions 87 vn CHAPTER III 89 POLYAMINES AND OXIDATIVE STRESS 89 Introduction 90 Materials and Methods 96 Glutathione Reductase (EC 1.6.4.2) Assay 96 Ascorbate Peroxidase (EC 1.11.1.11) Assay 97 Monodehydroascorbate Reductase (EC 1.6.5.4) Assay 97 Results 98 Total Soluble Protein 98 Standardization of Glutathione Reductase activity 99 Glutathione Reductase (GR) 99 Ascorbate Peroxidase (APX) 103 Monodehydroascorbate Reductase (MDHAR) 103 Glutathione 103 Accumulation of Ca and K 105 Mitochondrial Activity and Membrane Function 105 Discussion 107 Conclusions 116 CHAPTER IV 117 POLYAMINES AND ALUMINUM TOXICITY 117 Introduction 117 Materials and Methods 120 Cell growth and harvest 120 Results 121 Effect of Al and Ca on cell growth 121 Effect of Al and Ca on mitochondrial activity and membrane permeability 121 Effect of Al and Ca on cellular polyamines 127 Effect of Al and Ca on accumulation of inorganic ions 127 Effect of Al on GSH, y-EC and PC2 133 Effect of Al and Ca on cellular free amino acids 133 Discussion 143 Conclusions 152 Vlll CHAPTER V 153 POLYAMINES, GAD AND GABA 153 Introduction 153 Materials and Methods 159 Characterization of glutamate decarboxylase (GAD) activity 159 Results 160 Glutamate decarboxylase (GAD) 160 Effects of 3-MPA on growth and fresh weight of cells 160 Effects of 3-MPA on mitochondrial activity and membrane function 161 Effects of 3-MPA on polyamines, GABA, and glutathione 162 Discussion 162 Conclusions 171 CHAPTER VI 172 PUTRESCINE OVERPRODUCTION AND CARBON ASSUVHLATION 172 Introduction 173 Materials and Methods 175 14C sucrose feeding 175 14C arginine feeding 176 Results 176 I4C-sucrose feeding 176 14C arginine feeding 178 Discussion 178 Conclusions 181 CHAPTER VII. 183 PUTRESCINE OVERPRODUCTION AND THE PROLINE PATHWAY 183 Introduction 183 Materials and Methods 186 OAT assay 186 PCR amplification 187 TOPO-ligations and transformation of Escherichia coli 188 Plasmid DNA isolation 189 Analysis of ligated products by restriction digestion 189 ix DNA Sequencing and sequence analysis 189 RNA Extraction 190 cDNA synthesis 191 Normalized gene expression 191 Results 192 OAT activity and total protein content 192 Normalized gene expression 192 Celluar proline content 198 Discussion 198 Conclusions 200 GENERAL CONCLUSIONS 202 APPENDICES 208 APPENDIX A 209 A STRUCTURAL REPRESENTATION OF METABOLISM OF POLYAMINES AND RELATED AMINO ACIDS 209 APPENDIX B 210 ANALYSIS OF GENE EXPRESSION (Results of Dr. Andrew Page; Page et al., 2007) 210 APPENDIX C 215 EFFECTS OF Al AND Ca ON CELLULAR AMINO ACIDS 215 APPENDIX D 218 ENHANCED PUT METABOLISM AND CELLULAR INORGANIC IONS 218 LITERATURE CITED 223 x LIST OF TABLES Table 1. Partial list of genes of the polyamine metabolic pathway and the plants from which they have been cloned 11 Table 2. Partial list of genes of the ornithine and arginine biosynthetic pathway and the plants from which they have been cloned 13 Table 3. Partial list of genes of the proline biosynthetic pathway and the plants from which they have been cloned 15 Table 4. List of transgenic experiments in plants with genes encoding polyamine metabolic enzymes 18 XI LIST OF FIGURES Figure 1. Structures of the three common polyamines 1 Figure 2.