Extracellular matrix of the charophycean green algae

228 Pages·2015·1.81 MB·English

by  Sarah Nelson Kiemle

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Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's
Reports - Open Reports
2010
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Sarah Nelson Kiemle
Michigan Technological University
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Copyright 2010 Sarah Nelson Kiemle
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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.
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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 _______________________________________________________

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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.

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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
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RESULTS ____________________________________________________________________________ 58
Cellulose content ________________________________________________________________________ 58
Chemical composition of the insoluble residue ________________________________________________ 60
(cid:38)(cid:75)(cid:68)(cid:85)(cid:68)(cid:70)(cid:87)(cid:72)(cid:85)(cid:76)(cid:93)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:82)(cid:73)(cid:3)(cid:11)(cid:20)(cid:15)(cid:3)(cid:22)(cid:12)(cid:3)(cid:533)-glucan in the cell wall _____________________________________________ 62
(cid:11)(cid:20)(cid:15)(cid:3)(cid:22)(cid:12)(cid:3)(cid:533)-glucan localization in the cell wall ____________________________________________________ 64
DISCUSSION __________________________________________________________________________ 66
LITERATURE CITED _____________________________________________________________________ 72

CHAPTER 3: HEMICELLULOSIC POLYMERS IN THE CGA: UNIQUE
MIXED-LINKAGE (1 (cid:198)(cid:198) 3),(1 (cid:198) 4)-(cid:506)-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
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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 FLACCIDUM ___________________________________ 134
ABSTRACT __________________________________________________________________________ 134
INTRODUCTION _______________________________________________________________________ 135
METHODS __________________________________________________________________________ 138
Isolation and culture ____________________________________________________________________ 138
Defatting cell walls _____________________________________________________________________ 140
Fractionation of cell walls ________________________________________________________________ 140
Compositional assays ___________________________________________________________________ 141
Neutral sugar analysis ___________________________________________________________________ 141
Linkage analysis _______________________________________________________________________ 142
Monoclonal antibody labeling _____________________________________________________________ 143
RESULTS ___________________________________________________________________________ 143
Chlorokybus atmophyticus _______________________________________________________________ 144
Klebsormidium flaccidum ________________________________________________________________ 145
Spirogyra sp. EPS ______________________________________________________________________ 146
DISCUSSION _________________________________________________________________________ 150
LITERATURE CITED ____________________________________________________________________ 153

CHAPTER 6: THE EXTRACELLULAR POLYMERIC SUBSTANCES OF
DESMIDS (CONJUGATOPHYCEAE, STREPTOPHYTA): CHEMISTRY,
STRUCTURAL ANALYSES AND IMPLICATIONS IN WETLAND BIOFILMS.
____________________________________________________________________ 160
ABSTRACT __________________________________________________________________________ 160
INTRODUCTION _____________________________________________________________________ 162
METHODS __________________________________________________________________________ 165
Isolation and culture of desmids ___________________________________________________________ 165
Compositional assays ___________________________________________________________________ 167
Monosaccharide analysis _________________________________________________________________ 167
Linkage analysis _______________________________________________________________________ 168
Light microscopy, Confocal laser scanning microscopy and Lectin labeling ________________________ 169
Variable pressure scanning electron microscopy ______________________________________________ 170
RESULTS ___________________________________________________________________________ 170
EPS structure __________________________________________________________________________ 170
Components of Extracellular Matrix ________________________________________________________ 172
Polymer structure in biofilm ______________________________________________________________ 177
DISCUSSION _________________________________________________________________________ 179
LITERATURE CITED ____________________________________________________________________ 184

CHAPTER 7: CONCLUSIONS AND MAJOR FINDINGS _________________ 192
LITERATURE CITED ____________________________________________________________________ 198
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APPENDICES _______________________________________________________ 209
PROCEDURES FOR DEFATTING AND CLEANING CELL WALLS _________________________________________ 209
Alcohol Insoluble Residue protocol _________________________________________________________ 209
Methanolic KOH Extraction _______________________________________________________________ 209
CELL WALL FRACTIONATION ______________________________________________________________ 210
DERIVATIZATION FOR NEUTRAL SUGAR ANALYSIS ________________________________________________ 212
QUANTIFICATION OF MONOSACCHARIDE ANALYSIS ______________________________________________ 214
LINKAGE PATTERN ANALYSIS ______________________________________________________________ 216
METHYLATION ANALYSIS OF URONIC ACID OLIGOSACCHARIDES ______________________________________ 218
GCMS STARTUP AND SHUT DOWN _________________________________________________________ 219
UPDEGRAFF PROCEDURE FOR CELLULOSE _____________________________________________________ 220
COLORMETRIC ASSAYS __________________________________________________________________ 221
Phenol-Sulphuric _________________________________________________________________ 221
Carbazole Assay _________________________________________________________________ 222
DNS reducing sugar Assay _________________________________________________________ 223
DIGESTION FOR OLIGOSACCHARIDE MASS PROFILING _____________________________________________ 224
Digestion with Lichenase __________________________________________________________ 224
Digestion with xylanase M2 ________________________________________________________ 224
Digestion with endo-1,3-(cid:628)-D-glucanase _______________________________________________ 225
Preparation of MALDI –TOF –MS samples _____________________________________________ 225
Oligosaccharide Masses for Maldi-TOF Results _________________________________________ 226
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Acknowledgements

I could not have successfully completed this work without the encouragement,
support, and advice of many people. I would first like to thank my committee members:
Dr. David Domozych, Dr. Heather Youngs, and Dr. Stephen Hackney. I would also like
to especially thank Dr. Michael Gretz for his advice, direction, and consistent
encouragement. In fact, I would be remiss if I did not thank the entire Gretz family,
including Mrs. Anna Gretz and MicheleGretz, for always treating me like a member of
the family and for taking such wonderful care of me throughout my years in the Upper
Peninsula. I would also like to express appreciation to Dr. Kathy Domozych who
welcomed me into her home on multiple occasions.

My special thanks goes to the wonderful people in the Department of Biological
Sciences, including Patty Asselin and Emily Betterly for their unending help and
patience. Thank you to Jeff Lewin who spent many hours helping me build and fix
equipment and to Dr. Casey Huckins always listened when I needed to talk. Further, I
would like to express my heartfelt thanks to my wonderful lab mates: Dr. Brent
Bellinger, Dr. Melba Apoya-Horton, Dr. Abass Abdullahi, Dr. Ash Haeger, Dr. Utpal
Adhikari, and Erin McKenney. Thank you also to my outstanding undergraduate
students: Michelle Riccio, Holly Burger, Kate Pote, Jacob Jaszczak, Corey Momont, and
all other members of the Gretz Lab past and present.

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Lastly, the ultimate success of this interminable endeavor is certainly due to the
love and support of the incredible friends I have made here. Thank you especially to
Sandy Hubscher who has been my rock and support throughout in addition to being the
editor of this entire document, and thanks also to Sandy’s husband, Tim Eisele, and their
daughters, Samantha, and Rosemary, who are the best tension tamers. Finally, thank you
to Meagan Harless who is always able to talk me down.

“If it looks like a duck, and quacks like a duck, we have at least to consider the possibility
that we have a small aquatic bird of the family Anatidae on our hands.”
DDouglas Adams, (1952 - 2001)

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