Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Molecular tools for the identification and localization of algal cell wall components Author(s) Raimundo, Sandra Cristina Carneiro Publication Date 2016-01-11 Item record http://hdl.handle.net/10379/5454 Downloaded 2021-09-24T12:47:53Z Some rights reserved. For more information, please see the item record link above. National University of Ireland Galway School of Natural Sciences Molecular tools for the identification and localization of algal cell wall components A thesis submitted in partial fulfilment of the requirements for the degree of Doctor Philosophiae (PhD) Sandra Cristina Carneiro Raimundo January 2016 Dr. Zoë A. Popper Botany and Plant Science Ryan Institute for Environment, Marine and Energy Research School of Natural Sciences National University of Ireland Galway Galway Ireland Dr. Maria Tuohy Molecular Glycobiotechnology Group Department of Biochemistry School of Natural Sciences National University of Ireland Galway Galway Ireland Professor Azeddine Driouich Laboratoire Glycobiologie et Matrice Extracellulaire Normandie Université Institute for Research and Innovation in Biomedicine, Végétal, Agronomie, Sol, et Innovation 76821 Mont-Saint-Aignan France Professor Peter Dockery Anatomy School of Medicine National University of Ireland Galway Galway Ireland I want to acknowledge not only the people who contributed to the positive outcome of this thesis, but also, and above all, to the people who truly believed in me. I thank my supervisor Dr. Zoë A. Popper for the guidance, patience and help. I also thank Dr. Maria Tuhoy, Professor Azeddine Driouich, Professor Peter Dockery, Dr. Dagmar Stengel, Professor Charles Spillane, Síle Mhic Dhonncha, Pierce Lalor, and Dr. Kerry Thompson. My sincere acknowledgements to the collaborators that provided the equipment, tools and valuable scientific knowledge essential to the success of this project: Professor Michael G. Hahn, the members of his lab and of the CCRC, especially Christina Hopper; Professor David Domozych for his priceless help, and his wife Dr. Catherine Domozych. Thank you to my precious family. Words won´t be enough to express my gratitude to Udo Nitschke. This research was founded by the Science Foundation Ireland Research Frontiers Programme (grant 11/RFP/EOB/3345). Cell wall glycan-directed monoclonal antibodies (mAbs) are sensitive tools for the detection of wall components in vitro (glycome profiling) and in situ (immunolocalization). Although extensively applied to land plant and green microalgal research, their application to seaweeds is rare and mAbs generated against commercially valuable red- (carrageenans) and brown- (sulfated fucans and alginates) seaweed polysaccharides were never made available to the scientific community. Although brown seaweeds are only distantly related to land plants and the other algal groups, the glycome profile of Fucus vesiculosus indicated that several mAbs, belonging to a group that recognizes arabinogalactans (RG-I/AG), bound to extracts and gave distinct in situ labelling patterns. This led to the conclusion that epitopes from structurally different galactans are present and have distinct spatio-temporal distributions within seaweed tissues. The LAMP mAb, generated against laminaran, recognizes a (1,3)-- glucan epitope. While it is commonly used for the immunolocalization of callose in plants it has only rarely been applied to brown seaweeds. However, distinct in situ labelling patterns in F. vesiculosus and Laminaria digitata indicate that LAMP is a valuable probe for brown seaweed research. The LM7 mAb recognizes an epitope present in homogalacturonan, localized in land plants to the corners of the intercellular spaces between adherent and separated cell walls. In situ labelling of F. vesiculosus and L. digitata highlight this mAb as an excellent marker for alginates. These results show that some land plant cell wall glycan epitopes are present in brown seaweeds, providing new insights into cell wall evolution. Additionally identification of existing mAbs that can recognize epitopes present in seaweed cell walls has diversified and added valuable additional tools for algal research. The Vreeland mAbs, generated against seaweed polysaccharides, were tested to see if they remained viable after nearly 40 years storage. Some of the mAb supernatants indeed proved viable and can be used to produce hybridoma lines, creating antibodies for seaweed research. The green microalga Penium margaritaceum, used as a unicellular model organism for primary cell walls, was shown to be highly sensitive to plant hormones that had severe repercussions for growth and morphology. However, more pertinently, using mAbs, many of the impacts were seen to specifically involve the cell wall. These studies pave the way for future research regarding the restructuring of specific cell wall components, and wall architecture, by hormones as well as investigation of the mode of action of inhibitors. I List of Figures ....................................................................................................................... IX List of Tables ...................................................................................................................... XIII Abbreviations ...................................................................................................................... XV Publications and communications by the author .......................................................... XVII 1 General Introduction ..................................................................................................... 1 1.1. Evolution and diversity of land plants and algae ............................................ 3 1.2. Comparison between land plant and algal cell walls ...................................... 7 1.3. Plant cell walls................................................................................................ 8 1.4. Algal cell walls ............................................................................................. 11 1.4.1. Seaweed cell walls ........................................................................... 11 1.4.2. Charophycean green algal cell walls ............................................... 20 1.5. Penium margaritaceum as a model organism .............................................. 22 1.6. Monoclonal antibodies as tools for the study of cell walls ........................... 23 1.6.1. Monoclonal antibody production ..................................................... 24 1.6.2. Plant cell wall-glycan directed mAbs .............................................. 26 1.6.3. Seaweed cell wall-glycan directed mAbs ........................................ 27 1.7. Plant hormones ............................................................................................. 28 1.7.1. Plant hormones in algae ................................................................... 30 1.7.2. Interactions between plant hormones and cell walls ....................... 31 1.8. Research Objectives ..................................................................................... 33 2 Immunolocalization of cell wall carbohydrate epitopes in seaweeds: presence of land plant epitopes in Fucus vesiculosus L. (Phaeophyceae) ........................................... 37 2.1. Introduction .................................................................................................. 39 2.2. Materials and Methods ................................................................................. 44 2.2.1. Algal material .................................................................................. 44 2.2.2. Cell wall extraction .......................................................................... 44 2.2.3. Monosaccharide composition .......................................................... 45 2.2.4. Enzyme-Linked Immunosorbent Assay .......................................... 45 2.2.5. Light microscopy ............................................................................. 46 2.2.6. Monoclonal antibodies .................................................................... 46 III 2.2.7. Immunohistochemistry .................................................................... 52 2.3. Results ......................................................................................................... 54 2.3.1. Cell wall monosaccharide composition ........................................... 54 2.3.2. Glycome Profiling ........................................................................... 54 2.3.3. Immunolocalization ......................................................................... 57 2.4. Discussion .................................................................................................... 63 2.4.1. Glycome Profiling ........................................................................... 63 2.4.2. -Glucan epitope ............................................................................. 64 2.4.3. Pectin-associated epitopes ............................................................... 64 2.4.4. AG-3 mAb ....................................................................................... 66 2.5. Acknowledgements ...................................................................................... 68 3 Application of the LAMP monoclonal antibody as a molecular marker for the study of brown seaweed (Phaeophyceae) cell walls ..........................................................