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Alpha-Glucan, Water Dikinase and the Control of Starch Degradation in Arabidopsis Thaliana Leaves

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Alpha-Glucan, Water Dikinase and the Control of Starch Degradation in Arabidopsis Thaliana Leaves Alpha-Glucan, Water Dikinase and the Control of Starch Degradation in Arabidopsis thaliana Leaves Alastair William Skeffington A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy John Innes Centre Norwich September 2012 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there-from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. i Abstract This thesis examines the role of alpha-glucan, water dikinase (GWD) in the control of starch degradation in Arabidopsis thaliana leaves. Starch is degraded at night in Arabidopsis leaves to provide carbon for metabolism and growth. The rate of starch degradation is constant through the night and is precisely regulated such that starch reserves last until dawn. Despite the importance of this regulation for normal plant growth, we do not know which enzymes of the pathway are regulated to modulate flux. In this work I focus on the first enzyme in the starch degradation pathway, GWD. This enzyme phosphorylates the starch granule surface, thereby stimulating starch degradation by hydrolytic enzymes. It is hypothesised that GWD is important for the control of starch degradation. GWD shows large diel oscillations in transcript abundance. The enzyme is redox responsive in vitro, and is phosphorylated and SUMOylated in vivo. My work revealed that GWD protein levels are essentially invariant over the diel cycle. Studying plants in which GWD transcript was targeted by inducible RNAi and rapidly decreased to low levels revealed that GWD has half life of 2 days and a very low flux control coefficient for starch degradation. Taken together these findings demonstrate that large diel oscillations in GWD transcript abundance are not important for the daily control of starch degradation. Using transgenic plants in which native GWD was replaced with mutant forms of the enzyme, I demonstrate that redox responsive properties and certain SUMOylation and phosphorylation sites of GWD are not of in vivo relevance for appropriately timed starch degradation. Finally, I identify a previously unknown role for GWD in starch synthesis and find ROC4 to be a novel, putative GWD interaction partner. i Acknowledgements First, a massive thank you to my supervisor, Professor Alison Smith, for her constant enthusiasm: making starch (even more) exciting, for attentive supervision and for staying engaged through the more difficult times, before the projects began to bear fruit. I am grateful to the other members of my supervisory committee, Professor Caroline Dean and Professor Martin Howard for their invaluable advice and direction. I would also like to thank Professor Caroline Dean and Professor Phillip Poole for hosting me in their labs during the rotation year of my PhD. Both rotations were wonderful experiences from which I learnt a lot. Also thanks to Professor Nick Brewin, for his incredible dedication to making the rotation programme as valuable an experience as possible. Dr Sean Murray and Dr Richard Morris kindly gave their time to discuss the more quantitative aspects of this thesis. Their advice was indispensible. I am indebted to Professor Sam Zeeman and Professor Wilhelm Gruissem (ETH, Zurich) for allowing me to work in their labs for a month. While I was there I received help from many people, but would especially like to thank Dr Oliver Kötting, David Seung and Leonie Luginbühl for making me so welcome. Of course, it has been the people I have shared the lab with who have really made the experience over the past three years. In particular, I would like to express my admiration for Dr Marilyn Pike, who posses superhuman patience and irrepressible good cheer. Many thanks also to Doreen Feike, Dr Sam Mugford, Dr Vasilios Andriotis, Dr Cristina Pignocchi, Dr Alistair McCormick, Brendan Fahy, Dr Matilda Crumpton-Taylor, Dr Christian Ruzanski, Dr Sylviane Comparot-Moss, Dr Paul Barratt and Dr Thomas Howard for their help and advice and for making the lab a lovely place to work. Special thanks go to Dr Alex Graf, without whom much of my PhD would not have been possible. The John Innes Centre has been a great place to be a student, and that is down to the people I have shared my time with while here. In particular I would like to thank Ruth ii Bryant, Simon Lloyd, Philippa Borrill as well all the people I have shared 101 City Road with, for their friendship and many happy times. Where would I be without my friends from Cambridge: Natscis and Notscis? Few are privileged to have such a wonderful and close group of friends. Collectively and individually you have been a constant source of strength and joy and I look forward to many more wonderful times together. The last year has not always been easy, and to a large extent I owe my sanity to Wiebke Apel. Thank you so much for your incredible friendship, love and support. I have so many treasured memories of our time together already, and I know we have more wonderful times ahead of us. Finally, but most importantly, I have my family to thank for everything: for their encouragement and support through so many years of education, for their companionship, love and good humour. iii Table of Contents Abstract .............................................................................................................................. i Acknowledgements...........................................................................................................ii 1 Introduction ................................................................................................................ 1 1.1 Starch and plant growth ...................................................................................... 1 1.2 The synthesis of the starch granule .................................................................... 5 1.2.1 Starch structure........................................................................................ 5 1.2.2 The enzymes of starch synthesis in chloroplasts................................... 10 1.2.3 The control of starch synthesis .............................................................. 15 1.3 The enzymes of starch degradation .................................................................. 18 1.3.1 Attacking a crystalline granule ............................................................. 20 1.3.2 Degrading the granule ........................................................................... 24 1.3.3 Further metabolism in the stroma ......................................................... 28 1.3.4 The fate of maltose in the cytosol ......................................................... 29 1.4 The control of starch degradation ..................................................................... 30 1.4.1 The initiation of starch degradation ...................................................... 30 1.4.2 Regulation of starch degradation rates .................................................. 33 1.4.2.1 Predicting the time until dawn ............................................................ 36 1.4.2.2 Measuring starch ................................................................................. 37 1.4.2.3 Performing arithmetic division ........................................................... 39 1.5 Regulating the enzymes of starch degradation ................................................. 40 1.5.1 Properties of the genes and enzymes of starch degradation which might be relevant for control of the pathway .................................................. 41 1.5.1.1 Transcriptional regulation ................................................................... 41 1.5.1.2 Redox regulation ................................................................................. 42 1.5.1.3 Other post-translational modifications ............................................... 44 1.5.1.4 Regulation of protein localisation ....................................................... 45 1.5.1.5 Feedback regulation ............................................................................ 46 1.5.1.6 Protein- protein interactions ............................................................... 47 1.5.2 Other evidence pertinent to the regulation of starch degradation ......... 48 1.5.2.1 Evidence for the role of granule phosphorylation in the regulation of starch degradation ............................................................................... 48 1.5.2.2 The requirement for a starch granule .................................................. 49 iv 1.5.2.3 Thermodynamics ................................................................................ 50 1.5.3 AtGWD as a candidate for an enzyme regulated to modulate flux....... 51 1.6 Outline of experimental approach .................................................................... 53 2 Materials and Methods ............................................................................................. 54 2.1 Supplier details ................................................................................................. 54 2.2 Chemicals ......................................................................................................... 55 2.3 Enzymes ........................................................................................................... 55 2.4 Plant material ...................................................................................................
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