Research Collection Doctoral Thesis Post-Translational and Transcriptional Regulation of Storage Glucan Metabolism in Model and Non-Model Plants Author(s): Umhang, Martin Publication Date: 2011 Permanent Link: https://doi.org/10.3929/ethz-a-6618292 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH Nr. 19562 Post-Translational and Transcriptional Regulation of Storage Glucan Metabolism in Model and Non-Model Plants A dissertation submitted to the ETH ZÜRICH For the Degree of Doctor of Science Presented by Martin Umhang MSc in Biology, University of Bern, Switzerland Born the 20 th July, 1980 in Thun, Switzerland Accepted on the recommendation of Prof. Samuel C. Zeeman, examiner Prof. Julia Vorholt, co-examiner Dr Lee Sweetlove, co ‐‐‐examiner 2011 Acknowledgements I would like to thank Prof. S. C. Zeeman for giving me the opportunity to do my PhD in his lab. Sam has provided me with inputs for my scientific work and gave advice when I needed it. He gave me the possibility to explore exciting new areas in research and helped me to tackle the challenges. I am very grateful for the time I could work with him. I have certainly learned a lot; not only in scientific terms – thank you! I am thankful to Sebastian Streb; besides having helped a lot in the analyses of the C. peltata glucan structures and sugars, he always provided new ideas and helped to critically review results. My thanks go to Oliver Kötting; he has provided the SEX4 protein as well as the P-oligos I have been able to use. I am glad to see that he is following up some of my work. I am also very grateful to Sang- Kyu Lee, Michaela Stettler, Heike Reinhold and Sylvain Bischof; they helped in some of my experiments, did initial experiments or provided materials I could profit from. I have very much appreciated the work done by Simona Eicke, who did all the TEM work and helped in many other aspects of daily lab business. Our gardeners, Sabine Klarer and André Imboden, have been invaluable, taking care of my Arabdopsis or C. peltata plants. Weihong Qi, Marzanna Küenzli and Paolo Nanni from the FGCZ have helped a lot with either the C. peltata transcriptome sequencing or the mass spectrometry analyses. I am very thankful for all the work they have done to support my projects. Furthermore, I would like to thank all members of the ‘Plant Biochemistry’ group. I have spent a great time in the D36 and B33 labs. It was a pleasure to work with you, Carmen, Lilly or Ernst, just to name a few among many. I am grateful to Alex Graf and Jychian Chen; I have been able to use some of their results and materials for my thesis. My family and my friends have always supported me in my plans and projects – I am very thankful for the many relaxing hours; past or yet to come Finally, I feel very grateful to my girlfriend, Sonja Pfammatter. She encouraged me when things did not work well and she has shared my joy about my achievements. She had to share my attention with a time consuming PhD project but has always supported me in my dreams. Thank you! SUMMARY .......................................................................................................................... 1 ZUSAMMENFASSUNG ..................................................................................................... 3 ABBREVIATIONS .............................................................................................................. 5 1. INTRODUCTION ............................................................................................................... 7 1.1 Starch - A Vital Plant Product for Mankind ............................................................................. 7 1.2 Starch Is the Major Carbon Storage Compound in Plants ...................................................... 8 1.3 The Structure and Composition of Starch Granules ................................................................ 9 1.3.1 Starch Granule Morphology ........................................................................................................ 9 1.3.2 Starch Composition ................................................................................................................... 10 1.3.3 Starch Granule Architecture ...................................................................................................... 11 1.4 Starch Synthesis in Autotrophic and Heterotrophic Tissue ................................................... 13 1.5 The Synthesis of Amylopectin and Amylose ............................................................................ 17 1.5.1 Starch Synthases ........................................................................................................................ 17 1.5.2 Starch Branching Enzymes........................................................................................................ 18 1.5.3 Debranching Enzymes in Starch Synthesis ............................................................................... 19 1.5.4 Protein Complex Formation among Starch Synthesizing Proteins............................................ 20 1.5.5 The Architecture and Structure of Glycogen ............................................................................. 21 1.6 Factors Affecting the Crystallinity of the Starch Granule ..................................................... 22 1.7 Starch Degradation in Leaves ................................................................................................... 23 1.7.1 Starch Phosphorylation .............................................................................................................. 23 1.7.2 Degradation of Linear Chains in Starch Degradation ............................................................... 26 1.7.3 Debranching Enzymes in Starch Degradation ........................................................................... 27 1.7.4 Starch De-Phosphorylation ........................................................................................................ 28 1.7.5 Metabolism of Starch Breakdown Products .............................................................................. 28 1.8 Cecropia peltata , Glycogen Synthesis in a Vascular Plant ...................................................... 29 1.8.1 Cecropia peltata – A Many-Sided Subtropical Tree ................................................................. 29 1.8.2 Müllerian Bodies – Myrmecophytic Food Structures of Cecropia peltata ............................... 30 1.8.3 Glycogen Deposition in Müllerian Bodies ......................... Fehler! Textmarke nicht definiert. 1.9 Scope of the Work Presented .................................................................................................... 33 2. MATERIAL AND METHODS ........................................................................................ 34 2.1 Plant material ............................................................................................................................. 34 2.2 Standard SDS-PAGE and Western Blotting ........................................................................... 35 2.3 Native PAGE and native PAGE blotting ................................................................................. 35 2.4 Co-Immunoprecipitation of BAM1 and LSF1 ........................................................................ 36 2.5 Gel Filtration Chromatography ............................................................................................... 37 2.6 Chloroplast Isolation and Measurement of GAPDH and PEP-Carboxylase Activities...................................................................................................................................... 37 2.7 Carbohydrate Extraction and Measurements ......................................................................... 38 2.7.1 Quantification of Starch and WSP ............................................................................................ 39 2.7.2 Chain Length Distributions of Starch and Water Soluble Polyglucans ..................................... 39 2.8 High pH Anion Exchange Chromatography Coupled to Pulsed Amperometric Detection ..................................................................................................................................... 40 2.9 Plant Transformation and Complementation ......................................................................... 41 2.10 Tandem Affinity Purification and Myc-CoIP.......................................................................... 42 2.11 Mass-Spectrometric Analyses of Tandem-Affinity-Purified Protein Samples ..................... 43 2.12 Purification and Digestion of Phospho-Oligosaccharides ...................................................... 44 2.13 Digestion of Amylopectin Using BAM1-TAP( bam1 ) and BAM1-TAP( lsf1 ) ......................... 45 2.14 Measurement of β-Amylase Activity ........................................................................................ 45 2.15 Transmission Electron Microscopy of C. peltata leaves and Müllerian Bodies .................... 46 2.16 mRNAseq of C. peltata Leaves and Müllerian Bodies ............................................................ 46 2.16.1 C. peltata RNA Extraction, cDNA Synthesis and Normalization ............................................. 46 2.16.2 Library preparation for Illumina and 454 Pyrosequencing