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University of Cape Town The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University Sucrose Phosphate Synthase activity and gene expression in relation to dehydration induced sucrose accumulation in the resurrection plant Xerophyta humilis Zac Eliot McDonaldTown Cape of A thesis submitted for the Degree of University DOCTOR OF PHILOSOPHY Department of Molecular and Cell Biology University of Cape Town November 2008 ACKNOWLEDGEMENTS I would like to thank my supervisors, Assoc. Prof. Nicola Illing and Prof. Jill Farrant. Their insights, expert advice and encouragement ensured that I reached my research goals. I have benefitted greatly from their extensive experience and am grateful for the time and effort they have invested in me. To all the Illing lab members and technical staff, past and present, I am sincerely thankful for all your advice and support. A special note of thanks is extended to Dr Aubrey Shoko who generously gave of his time and knowledge and to Faezah Davids for her technical assistance in general lab matters. Town Dr Wolfram Weckwerth for giving me the opportunity to work in his group at the Max Planck institute of Plant Molecular Physiology, Potsdam, Germany.Cape A special thanks is extended to Dr Stefanie Wienkoop for her guidance with proteinof quantific ation and to Dr Katja Morgenthal for her invaluable assistance with the metabolite data analysis. I would also like to thank all the members of AG Weckwerth for their advice and for making me feel welcome during my stay in Gerrmany. Finally, I would like toUniversity thank my family and friends. This work would not have been completed without your encouragement and support. Thanks for sharing in the joy and for smoothing over the rough parts. I also greatly appreciate the financial support of the UCT EDP programme which allowed me to conduct my research. LIST OF ABBREVIATIONS............................................................................................................. VII LIST OF FIGURES ........................................................................................................................... X ABSTRACT .............................................................................................................................. XII CHAPTER 1 INTRODUCTION ................................................................................................. 1 Dehydration-induced sucrose accumulation in resurrection plants ......................................... 2 Function of sucrose accumulation in desiccation tolerance .................................................... 3 Role of SPS in regulating sucrose accumulation ..................................................................... 6 Regulation of SPS in photosynthetic tissue ............................................................................. 9 SPS activity and desiccation tolerance .................................................................................. 11 AIMS .......................................................................................................................................... 12 CHAPTER 2 SUCROSE ACCUMULATION AND METABOLITE CHANGES IN RESPONSE TO DEHYDRATION ........................................................................................... 13 INTRODUCTION ........................................................................................................................... 13 Sugar accumulation in orthodox seeds .................................................................................. 13 Sugar accumulation in angiosperm resurrection plants .........................................................Town 14 Function of sugar accumulation in desiccation tolerance ...................................................... 16 Sugar accumulation in desiccation sensitive plants ............................................................... 18 Carbon sources for sugar accumulation.................................................................................Cape 19 AIMS .......................................................................................................................................... 20 METHODS ..............................................................................................................................of ..... 21 Plant material ......................................................................................................................... 21 Determination of relative water content (RWC) ................................................................... 21 Efficiency of photosystem II electron transport (Φ PSII) ..................................................... 21 Leaf sampling strategy .......................................................................................................... 22 Processing and RWC estimation of harvested leaf tissue..................................................... 23 Sucrose, glucose Universityand fructose quantification ........................................................................ 25 Starch quantification .............................................................................................................. 25 Metabolite analysis ................................................................................................................ 26 Extraction procedure and sample preparation .................................................................... 26 GC-TOF-MS analysis ........................................................................................................ 27 RESULTS ..................................................................................................................................... 30 Changes in sucrose content ................................................................................................... 31 i Carbon sources supporting sucrose accumulation ................................................................. 34 Photosynthesis .................................................................................................................... 34 Carbohydrate carbon sources ............................................................................................. 34 Changes in metabolite profile during dehydration ................................................................ 35 Un-targeted metabolite analysis ......................................................................................... 35 Targeted metabolite analysis: changes in identified metabolites ....................................... 37 DISCUSSION ................................................................................................................................ 43 Changes in sucrose content during dehydration .................................................................... 43 Carbon sources for sucrose accumulation ............................................................................. 45 Changes in metabolite profile during dehydration ................................................................ 48 Conclusion ............................................................................................................................. 52 CHAPTER 3 SUCROSE PHOSPHATE SYNTHASE ACTIVITY IN RESPONSE TO DEHYDRATION ...................................................................................................................Town ..... 54 INTRODUCTION ........................................................................................................................... 54 SPS regulation in photosynthetics tissue ............................................................................... 54 Accurate assay of SPS activity ..............................................................................................Cape 57 Fluctuations in SPS activity ................................................................................................... 59 SPS activity and desiccation tolerance ..................................................................................of 60 AIMS .......................................................................................................................................... 60 METHODS ................................................................................................................................... 61 Plant material ......................................................................................................................... 61 Determination of relative water content (RWC) ................................................................... 61 SPS activity ........................................................................................................................... 61 Harvesting andUniversity processing of leaf tissue ............................................................................ 61 Protein extraction from freeze dried tissue for SPS assay ................................................. 62 Protein extraction from non-freeze dried tissue for SPS assay (for comparison experiment) ............................................................................................................................................ 62 SPS activity assay procedure ............................................................................................
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