Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's Reports - Open Reports 2010 Extracellular matrix of the charophycean green algae Sarah Nelson Kiemle Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons Copyright 2010 Sarah Nelson Kiemle Recommended Citation Kiemle, Sarah Nelson, "Extracellular matrix of the charophycean green algae", Dissertation, Michigan Technological University, 2010. https://doi.org/10.37099/mtu.dc.etds/190 Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biology Commons The Extracellular Matrix of the Charophycean Green Algae By Sarah Nelson Kiemle A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOLOGICAL SCIENCES, MICHIGAN TECHNOLOGICAL UNIVERSITY 2010 Copyright © Sarah Nelson Kiemle, 2010. All rights reserved. 1 This dissertation, "The Extracellular Matrix of the Charophycean Green Algae,” is hereby approved in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the field of Biological Sciences. DEPARTMENT: Biological Sciences Signatures: Dissertation Advisor: _________________________________________ Michael R. Gretz Department Chair _____________________________________________ K. Michael Gibson Date _______________________________________________________ 2 This work is dedicated in loving memory of Josephine Getty and Madison Kiemle, who always supported me in all my endeavors; also to my wonderful parents, Dee and Fred, and my sister, Kate, who have sustained my education and always encouraged me to pursue my love of science. 3 Table of Contents ACKNOWLEDGEMENTS ______________________________________________ 8 ABSTRACT __________________________________________________________ 10 LIST OF FIGURES ___________________________________________________ 12 LIST OF TABLES ____________________________________________________ 14 LIST OF ABBREVIATIONS ___________________________________________ 15 CHAPTER 1: ALGAL EXTRACELLULAR MATRIX TO LAND PLANT CELL WALLS _____________________________________________________________ 18 CHAROPHYCEAN GREEN ALGAE _____________________________________________________________ 18 FORMATION AND DIVERSIFICATION OF CELL WALLS _______________________________________________ 24 EVOLUTION OF PLANT CELL WALLS THROUGH CELL WALL POLYMERS. ___________________________________ 25 Cellulose ________________________________________________________________________ 26 Cross-linking glycans _______________________________________________________________ 28 Xyloglucans ____________________________________________________________________________ 28 Mixed-linkage glucans ___________________________________________________________________ 29 Xylan _________________________________________________________________________________ 30 Pectin ___________________________________________________________________________ 31 Homogalacturonan _______________________________________________________________________ 32 Rhamnogalacturonan I ____________________________________________________________________ 33 Rhamnogalacturonan II ___________________________________________________________________ 34 Beta- (1,3) glucans ________________________________________________________________ 34 Cell Wall Proteins _________________________________________________________________ 36 CONCLUSIONS ________________________________________________________________________ 37 OBJECTIVES ________________________________________________________ 39 LITERATURE CITED _____________________________________________________________________ 40 CHAPTER 2: IMPLICATIONS FOR POLYMER EVOLUTION OF BETA- GLUCANS IN THE CHAROPHYCEAN GREEN ALGAE __________________ 48 ABSTRACT ___________________________________________________________________________ 48 INTRODUCTION ________________________________________________________________________ 49 METHODS ___________________________________________________________________________ 52 Organism and growth conditions ___________________________________________________________ 52 Defatting of the cell walls _________________________________________________________________ 53 Cellulose content ________________________________________________________________________ 53 Fractionation of cell walls _________________________________________________________________ 54 Neutral sugar analysis ____________________________________________________________________ 54 Linkage analysis ________________________________________________________________________ 55 Enzymatic digestion _____________________________________________________________________ 55 Oligosaccharide characterization ___________________________________________________________ 56 Monoclonal antibody labeling ______________________________________________________________ 57 4 RESULTS ____________________________________________________________________________ 58 Cellulose content ________________________________________________________________________ 58 Chemical composition of the insoluble residue ________________________________________________ 60 &KDUDFWHUL]DWLRQRI ȕ-glucan in the cell wall _____________________________________________ 62 ȕ-glucan localization in the cell wall ____________________________________________________ 64 DISCUSSION __________________________________________________________________________ 66 LITERATURE CITED _____________________________________________________________________ 72 CHAPTER 3: HEMICELLULOSIC POLYMERS IN THE CGA: UNIQUE MIXED-LINKAGE (1 Æ 3),(1 Æ 4)-Ǻ-D-GLUCAN STRUCTURE ____________ 78 ABSTRACT ___________________________________________________________________________ 78 INTRODUCTION ______________________________________________________________________ 79 METHODS ___________________________________________________________________________ 81 Organism and growth conditions ___________________________________________________________ 81 Mechanical and chemical isolation of cell walls _______________________________________________ 82 Fractionation of cell walls _________________________________________________________________ 82 Neutral sugar analysis ____________________________________________________________________ 83 Linkage analysis ________________________________________________________________________ 83 Monoclonal antibody labeling ______________________________________________________________ 84 Enzymatic Digestion _____________________________________________________________________ 85 Reducing sugar assays ____________________________________________________________________ 86 Oligosaccharide characterization ___________________________________________________________ 86 RESULTS ____________________________________________________________________________ 87 Mixed-linkage oligosaccharide composition __________________________________________________ 87 MLG localization ________________________________________________________________________ 91 Characterization of hemicellulosic fractions ___________________________________________________ 92 DISCUSSION __________________________________________________________________________ 96 LITERATURE CITED ____________________________________________________________________ 102 CHAPTER 4: HOMOLOGOUS LAND PLANT PECTINS IN THE CHAROPHYCEAN GREEN ALGAE: PENIUM MARGARITACEUM, COSMARIUM TURPINI, CHLOROKYBUS ATMOPHYTICUS, AND KLEBSORMIDIUM FLACCIDUM ______________________________________ 107 ABSTRACT __________________________________________________________________________ 107 INTRODUCTION _______________________________________________________________________ 108 METHODS __________________________________________________________________________ 111 Isolation and culture ____________________________________________________________________ 111 Defatting of the cell walls ________________________________________________________________ 111 Fractionation of cell walls ________________________________________________________________ 112 Compositional assays ___________________________________________________________________ 112 Neutral sugar analysis ___________________________________________________________________ 113 Linkage analysis. _______________________________________________________________________ 113 Analytical methods _____________________________________________________________________ 114 Monoclonal antibody labeling _____________________________________________________________ 114 HPAEC-PAD System ___________________________________________________________________ 115 RESULTS ___________________________________________________________________________ 116 Immunochemical localization _____________________________________________________________ 116 Chemical composition ___________________________________________________________________ 117 Structural Analysis of HG ________________________________________________________________ 120 5 Evidence of other polymers in the cell walls of the CGA _______________________________________ 121 DISCUSSION _________________________________________________________________________ 124 LITERATURE CITED ____________________________________________________________________ 127 CHAPTER 5: AGP-LIKE POLYMER IN CHLOROKYBUS ATMOPHYTICUS CELL WALL AND SPIROGYRA SP EPS AND AN UNIQUE ALGAL POLYMER IN KLEBSORMIDIUM