STUDIES ON THE ROLES AND REGULATION OF TWO GLYOXYLATE REDUCTASES IN PLANTS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by WENDY LYNNE ALLAN In partial fulfilment of requirements for the degree of Doctor of Philosophy December, 2008 © Wendy Lynne Allan, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-50115-3 Our file Notre reference ISBN: 978-0-494-50115-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Canada ABSTRACT STUDIES ON THE ROLES AND REGULATION OF TWO GLYOXYLATE REDUCTASES IN PLANTS Wendy Lynne Allan Advisor: University of Guelph, 2008 Dr. Barry J. Shelp Succinic semialdehyde (SSA) is a mitochondrial intermediate in the metabolism of GABA, whereas glyoxylate is a peroxisomal metabolite generated from photorespiratory glycolate. Recent evidence indicates that distinct cytosolic and plastidial glyoxylate reductase isoforms from Arabidopsis thaliana (L.) Heynh (AfGLYRl and AfGLYR2, respectively) catalyse the in vitro NADPH-dependent conversion of SSA to gamma- hydroxybutyrate (GHB) and glyoxylate to glycolate. In this thesis, recombinant Arabidopsis GLYR1 was demonstrated to simultaneously catalyze both reactions in vitro. Time-course experiments revealed that GHB accumulated in leaves of Arabidopsis subjected to salinity, drought, submergence, cold or heat stress, and tobacco (Nicotiana tabacum L.) subjected to submergence. This was generally accompanied by higher GABA and Ala levels, higher NADPH/NADP+ ratios, and lower Glu levels. Furthermore, expression analysis of Arabidopsis revealed that the relative abundance of GLYR1 transcript was enhanced under salinity, drought, submergence, cold and heat, as was the GLYR2 transcript under cold and heat. Other time-course experiments revealed that Arabidopsis glyrl and glyr2 knockout mutants, unlike wild-type (wt) plants, did not have elevated levels of GLYR1 or GLYR2 transcript, GHB and NADPH in rosette leaves of plants subjected to submergence or low CO2 conditions (75 versus 380 [xmol mol"1 at 21% O2). Glycolate, glyoxylate and succinate also accumulated in glyrl and glyrl under low CO2, whereas Gly did not. Manipulation of redox balance via the use of Arabidopsis NAD kinase (NADK) mutants (nadkl and nadk3 knockout mutants, NADK1 overexpressor, and NADK2 underexpressor) altered the plant response to submergence. Thus, it can be concluded that: 1) GHB accumulation is a general response to abiotic stress and is regulated by both biochemical and transcriptional processes; and 2) GLYR activity is involved in the detoxification of both SSA and glyoxylate, and the recycling of phosphorylated pyridine nucleotides, thereby linking GABA metabolism and photorespiration to redox homeostasis. ACKNOWLEDGEMENTS Many people have contributed to the development of this thesis. Firstly, I would like to give thanks to Dr. Barry Shelp for his unwavering support, guidance and expertise during my tenure as a PhD student in his laboratory. He largely contributed to my enthusiasm and determination to succeed and has been a continuing source of inspiration to me. I would also like to thank my committee members Dr. David Wolyn, Dr. Chris Hall, Dr. Alan Bown and Dr. Barry Micallef for their advice, expertise and on-going support with a special thanks to Dr. Wolyn for his thoughtful comments on experimental design and statistical analysis. As well, I extend my gratitude to Dr. Katerina Jordon and to Dr. Bernie Grodinski for their generosity in allowing me the use of their lab equipment from time to time. I am also appreciative of the advice and help of greenhouse personnel and to the technical expertise and advice of many technicians and postdocs, including Alice Barker, Rodger Tschanz, Owen Van Cauwenberghe, Amina Mahkmoudova, and Gordon Hoover. I had many years of enjoyment and laughs with my labmates and cohorts and I am very much appreciative of their technical help, scientific debates, and how they regularly boosted my morale. I want to especially thank Shawn Clark and Jeff Simpson for their support and the extra help they provided me when I really needed it. Lastly, I would like to extend my love and gratitude to my husband and son, George who have endured my long working hours, and my stress-filled moments over the years. You have both been patient and it is now finally finished. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi CHAPTER 1 - INTRODUCTION 1 CHAPTER 2 - LITERATURE REVIEW 7 2.1. Metabolism and Toxicity of Aldehydes 7 2.1.1. Plant aldehydes 9 2.1.1.1. Detoxification of plant aldehydes 10 2.1.1.2. Aldehyde Dehydrogenases 11 2.1.1.3. Aldehyde Reductases 15 2.2. GABA Metabolism 17 2.2.1. Regulation 20 2.2.1.1 Glu decarboxylase (GAD) 20 2.2.1.2. y-aminobutyrate transaminase (GABA-T) 23 2.2.1.3. Succinic semialdehyde dehydrogenase (SSADH) 25 2.2.1.4. Glyoxylate/succinate semialdehyde reductase (GLYR) 26 2.2.2. Potential Roles 28 2.3. Pyridine Nucleotide Metabolism and Distribution 36 u 2.3.1. NAD+/NADH 37 2.3.2. NADP+/NADPH 41 2.4. Glyoxylate and Photorespiration 45 2.5 Stress and Photorespiration 50 CHAPTER 3 - y-HYDROXYBUTYRATE ACCUMULATION IN ARABIDOPSIS AND TOBACCO PLANTS IS A GENERAL RESPONSE TO ABIOTIC STRESS: PUTATIVE REGULATION BY REDOX BALANCE AND GLYOXYLATE ISOFORMS 53 3.1. Contribution 53 3.2. Acknowledgements 53 3.3. Abstract 54 3.4. Introduction 55 3.5. Materials and Methods 56 3.5.1. Growth of plant material 56 3.5.2 Arabidopsis exposure to drought, salinity, submergence, cold or heat 57 3.5.3. Tobacco submergence and recovery 58 3.5.4. Extraction and analysis of GHB, amino acids, and phosphorylated pyridine nucleotides 59 3.5.5 Expression and analysis of GLYR1 and GLYR2 in Arabidopsis 59 3.6. Results 60 in 3.6.1. Stress responsiveness of metabolites and phosphorylated pyridine nucleotides in Arabidopsis and tobacco 61 3.6.2 Recovery of GHB and related metabolites, and phosphorylated pyridine nucleotides in tobacco after removal from submergence 64 3.6.3. Stress responsiveness of GLYR transcripts in Arabidopsis 66 3.7. Discussion 66 3.7.1. Impact of abiotic stress on GAB A, Ala and redox levels 66 3.7.2 GHB accumulation is a general response to abiotic stress 72 3.7.3 Recovery of metabolite and redox levels after exposure to abiotic stress 74 3.7.4 Role of GLYR isoforms in detoxification of SSA and glyoxylate 75 3.7.5 Summary 77 CHAPTER 4 - REDOX BALANCE AND DETOXIFICATION OF SUCCINIC SEMIALDEHYDE AND GLYOXYLATE IN ARABIDOPSIS PLANTS SUBJECTED TO ENVIRONMENTAL CONDITIONS THAT PROMOTE y-AMINOBUTYRATE METABOLISM OR PHOTORESPIRATION 79 4.1. Contributions 79 4.2. Acknowledgements 80 4.3. Abstract 80 4.4. Introduction 81 4.5. Materials and Methods 83 iv 4.5.1. In vitro assay of simultaneous substrate utilization by recombinant Arabidopsis GLYR1 83 4.5.2. Generation and description of transgenic Arabidopsis lines 84 4.5.3. Growth of Arabidopsis 87 4.5.4 Analysis of GLYR and NADK expression in Arabidopsis 87 4.5.5. Measurement of enzyme activity in wild-type Arabidopsis and mutant lines 88 4.5.6. Exposure of wild-type Arabidopsis and mutant lines to submergence 90 4.5.7. Exposure of wild-type Arabidopsis and glyr mutants to photorespiratory conditions 91 4.5.8 Extraction and analysis of GHB, amino acids, organic acids and pyridine nucleotides 92 4.6. Results 93 4.6.1 Simultaneous utilization of succinic semialdehyde and glyoxylate by recombinant GLYR 1 93 4.6.2 GLYR activity in cell-free extracts from leaves of wild-type Arabidopsis and glyr mutants 95 4.6.3 Response of wild-type Arabidopsis and glyr mutants to submergence 97 4.6.4 Response of wild-type Arabidopsis and NADK mutants to submergence.... 103 4.6.5. Response of wild-type Arabidopsis and glyr mutants to varying CO2/O2 ratios 110 4.7. Discussion 120 v 4.7.1. Redox homeostasis and detoxification of succinic semialdehyde in Arabidopsis plants exposed to submergence 120 4.7.2. Redox homeostasis and detoxification of succinic semialdehyde and glyoxylate in Arabidopsis plants exposed to photorespiratory conditions 122 4.7.3.