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Rch Granule Number and Size in Arabidopsis Thaliana Leaves

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Rch Granule Number and Size in Arabidopsis Thaliana Leaves Starch granule number and size in Arabidopsis thaliana leaves Matilda Crumpton -Taylor A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy John Innes Centre Norwich September 2010 © This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived there from, may be published without the author ’s prior, written consent. Abstract The aim of my work was to understand the variation of starch granule number and size in Arabidopsis thaliana leaves. I describe first the development of methods to measure the number of starch granules per chloroplast. My work reveals that at a given developmental stage the number of granules per unit volume of stroma is much more conserved than the number of granules per chloroplast. In this study I explore the role of STARCH SYNTHASE4 (SS4) in granule initiation and the control of granule numbers, by analysing the phenotype of the Arabidopsis ss4 mutant. My results support and extend the observation that SS4 is required for starch synthesis in young leaves, and provide new information about its influence on the starch phenotype of the whole plant. In the absence of SS4, synthesis of glucan polymers as a whole seems to be blocked in the young leaves, indicating that SS4 may be responsible for the production of a small malto-oligosaccharide that is required as a primer for the polymerising activity of the other starch synthases and starch branching enzymes. I describe the use of the accumulation and regulation of chloroplast (arc ) mutants of Arabidopsis to explore the possibility that components of the chloroplast division apparatus could play an important role in granule initiation and/or starch metabolism. My results show that mutations in the chloroplast division components ARC3, ARC5, ARC6, At FtsZ1-1, At MinD1 and At MinE1 are not important and/or involved in starch metabolism. Taken as a whole my results show that starch granule number within Arabidopsis chloroplasts is related to stromal volume. I suggest that the most likely mechanism of granule initiation is via spontaneous crystallisation when the concentration of suitable glucans or malto-oligosaccharides within a volume of stroma reaches a critical level. I Table of Contents Abstract .............................................................................................................................. I List of Figures ............................................................................................................... VII List of Tables.................................................................................................................... X Acknowledgements ...................................................................................................... XIII Abbreviations ............................................................................................................... XIV 1 Introduction ............................................................................................................... 1 1.1 Aim ..................................................................................................................... 1 1.2 Starch Synthesis and Structure ........................................................................... 3 1.2.1 How starch is synthesised ........................................................................... 3 1.2.2 Control of starch synthesis .......................................................................... 8 1.2.3 Starch degradation and its control ............................................................. 11 1.2.4 The structure of starch ............................................................................... 15 1.3 Starch Granule and Glucan Chain Initiation .................................................... 19 1.3.1 Genetic Controls........................................................................................ 19 1.3.2 Physical Controls ...................................................................................... 30 1.3.3 Other possible controls .............................................................................. 32 1.4 Controls over starch granule number and morphology .................................... 33 1.4.1 The environment within the plastid........................................................... 36 1.4.2 Potential mechanisms for sensing leaf starch content .............................. 38 1.5 Justification for experimental approach ........................................................... 40 2 Materials and Methods ............................................................................................ 42 2.1 Plant Material ................................................................................................... 42 2.2 Growth conditions ............................................................................................ 42 2.2.1 Plant tissue culture ......................................................................................... 42 2.2.2 Plant growth conditions ................................................................................. 43 2.3 Bacteria and yeast strains ................................................................................. 43 2.4 Media and antibiotics ....................................................................................... 44 II 2.5 Cloning vectors ................................................................................................. 45 2.6 Oligonucleotides ............................................................................................... 46 2.7 General Molecular Methods ............................................................................. 47 2.7.1 Isolation of DNA ....................................................................................... 47 2.7.2 Polymerase chain reaction (PCR) ............................................................. 48 2.7.3 Genotyping of mutants .............................................................................. 48 2.7.4 Agarose gel electrophoresis ...................................................................... 49 2.7.5 DNA sequencing ....................................................................................... 49 2.7.6 RNA extraction ......................................................................................... 50 2.7.7 cDNA synthesis ......................................................................................... 50 2.7.8 Semi-quantitative reverse transcription polymerase chain reaction (RT- PCR) 51 2.7.9 Protoplast isolation .................................................................................... 52 2.7.10 Chloroplast isolation ................................................................................. 52 2.7.11 Preparation of protein extracts for SDS-PAGE ........................................ 53 2.7.12 Western blot analysis ................................................................................ 53 2.7.13 Gateway Cloning ....................................................................................... 54 2.8 Measurement of starch content ......................................................................... 56 2.8.1 Enzymes and Chemicals ........................................................................... 56 2.8.2 Extraction of starch from Arabidopsis leaves ........................................... 56 2.8.3 Enzymatic digestion of starch and phytoglycogen.................................... 57 2.8.4 Glucose Assay ........................................................................................... 57 2.9 Transformation of living cells .......................................................................... 58 2.9.1 Transformation of Escherichia coli .......................................................... 58 2.9.2 Transformation of Agrobacterium ............................................................ 58 2.10 Agrobacterium mediated transformation of Arabidopsis by floral dipping . 59 2.11 Cross pollination of Arabidopsis plants ........................................................ 59 2.12 Determination of luciferase activity ............................................................. 60 III 2.13 Curvature and growth studies ....................................................................... 60 2.14 Phenotypic analysis....................................................................................... 61 2.14.1 Purification of starch granules .................................................................. 61 2.14.2 Light microscopy and Transmission Electron Microscopy (TEM) .......... 62 2.14.3 Scanning Electron Microscopy (SEM) ..................................................... 62 2.14.4 Starch Granule measurements ................................................................... 63 2.14.5 Haemocytometer measurements ............................................................... 64 2.14.6 Iodine staining of rosettes and leaves........................................................ 65 2.14.7 Focused-Ion-Beam Scanning Electron Microscopy (FIB-SEM) .............. 65 2.15 Software Tools - Band quantification ........................................................... 65 2.16 Statistical analysis of significance ................................................................ 65 3 The relationship between starch granules
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