In situ analysis of grass cell wall polysaccharides By Jie Xue Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences November 2013 This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2013 The University of Leeds and Jie Xue The candidate confirms that all the work submitted in this thesis is her own. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Some of the work presented in Chapters 3 and 5 appear in the following publication: Jie Xue, Maurice Bosch, and J. Paul Knox, 2013. Heterogeneity and glycan masking of cell wall microstructures in the stems of Miscanthus x giganteus, and its parents M. sinensis and M. sacchariflorus. Plos ONE 8, e82114. “Happy is the man who is living by his hobby” George Bernard Shaw, 1856-1950 Acknowledgements Firstly I am very grateful to my supervisor, Professor Paul Knox, gave me a chance here for my PhD study, and also for all his help, instructions, guidance and patience for my study. The knowledgeable man (responsible for my interest in cell walls) taught me how to do an excellent cell wall research, how to produce a good scientific work and how to be a responsible scientist. Susan Marcus, the most kind woman in the Knox lab, for being taking very good care of me during these past three years, and also for her excellent lab techniques. I would also like to thank Dr Kieran Lee for his help with the exciting scientific ideas and also his experimental help. I am also grateful to my secondary supervisor, Professor Jurgen Denecke, for his help with the experiment techniques and scientific suggestions. My current and former Knox Cell Wall Lab members: Dr Tom Benians, Thomas Torode, Valérie Cornuault, Mercedes Hernández Gómez, Craig Deakin and Professor Carem Gledes Vargas-Rechia for giving me the passionate friendship in our office and lab. Martin Fuller, for his expertise in teaching me how to produce high quality resin embedded sections of grasses. Lance Penketh, for his kind help on using the plant growth facilities in University of Leeds greenhouse. I acknowledge the financial support from the University of Leeds and the China Scholarship Council over the past three years of my PhD. Finally, I would like to give my special thanks to my parents, my dearest father and mother who always stand by me, and give me spiritual support and so many encouragements during my study in Leeds University. i Abstract Plant cell walls are important resources of biomass and renewable energy. Miscanthus species are fast growing grasses that had been suggested to be potential bioenergy crops. Miscanthus x giganteus (M. x giganteus) grows faster and taller than its parents - M. sacchariflorus and M. sinensis. Wheat is the major food crop for human beings. Brachypodium distachyon is considered to be a model plant as it has a small genome size and rapid life cycle. In this study, cell wall polysaccharides in stems of the five grass species at different growth stages have been determined using an indirect immunofluorescence method with sets of specific monoclonal antibodies. Heteroxylan and mixed-linkage-glucan (MLG) epitopes are abundant in all grass species, but with different distributions in parenchyma regions and the detection levels of both polymers change during growth development. For M. x giganteus, pectic homogalacturonan (HG) epitopes were restricted to intercellular spaces of parenchyma and the high methylesterified LM20 HG epitope was strongly abundant in pith parenchyma cell walls. Some cell wall probes cannot access their target polymers because of masking by other polysaccharides. For all grass species in certain cell wall regions, the xyloglucan epitope is masked by heteroxylan, the pectic-galactan epitope is masked both by heteroxylan and MLG, and the LM2 AGP epitope is masked by pectin. These findings indicate the heterogeneity of polysaccharide distributions and molecular architectures in grasses, and this might be linked to grass anatomical and growth differences and may also be related to distinct cell wall functions between grass species. In summary, this fundamental knowledge of cell wall polysaccharide architectures in these important grass species will help us understand grass growth mechanisms, and may also be beneficial for the development of potential strategies for the efficient deconstruction of grass biomass that may make contribution to the biofuel industry. ii Contents Acknowledgements ................................................................................................. i Abstract .................................................................................................................. ii Contents ................................................................................................................. iii List of Figures ..................................................................................................... viii List of Tables ........................................................................................................ xv List of Abbreviations .......................................................................................... xvi Chapter 1 ................................................................................................................ 1 Introduction ........................................................................................................... 1 1.1 Importance of grasses ................................................................................ 2 1.2 Importance of plant cell walls ................................................................... 3 1.3 Plant cell wall polysaccharides ................................................................. 3 1.3.1 Cellulose ......................................................................................... 5 1.3.2 Hemicelluloses ............................................................................... 8 1.3.3 Pectic polysaccharides .................................................................. 17 1.4 Arabinogalactan-proteins (AGPs) ........................................................... 25 1.4.1 Structure ....................................................................................... 25 1.4.2 Classification ................................................................................ 27 1.4.3 Biosynthesis ................................................................................. 28 1.4.4 Function ........................................................................................ 29 1.5 Lignin ...................................................................................................... 30 1.5.1 Structure ....................................................................................... 30 1.5.2 Biosynthesis ................................................................................. 30 1.5.3 Function ........................................................................................ 31 1.6 Plant taxonomy & distinctive cell wall biochemistry of grasses ............ 31 1.6.1 Taxonomy of plants ...................................................................... 31 1.6.2 Plant cell wall biochemistry ......................................................... 33 1.7 Plant species studied in this thesis ........................................................... 35 1.7.1 Miscanthus species ....................................................................... 35 iii 1.7.2 Wheat ........................................................................................... 38 1.7.3 Brachypodium distachyon ............................................................ 39 1.8 Biofuel and biomass degradation ............................................................ 42 1.9 Cell wall probes ....................................................................................... 43 1.10 Summary ............................................................................................... 47 1.11 Aims and objectives .............................................................................. 47 Chapter 2 .............................................................................................................. 49 Materials and Methods ....................................................................................... 49 2.1 Plant materials ......................................................................................... 50 2.1.1 Miscanthus species ....................................................................... 50 2.1.2 Wheat ........................................................................................... 50 2.1.3 Brachypodium distachyon ............................................................ 50 2.2 Molecular probes ..................................................................................... 50 2.2.1 Monoclonal Antibodies ................................................................ 50 2.2.2 Carbohydrate-binding modules .................................................... 52 2.3 Microscopy and immunolabelling procedures ........................................ 52 2.3.1 Preparation of plant materials for microscopy ............................. 52 2.3.2 Steedman’s wax-embedding of plant materials and sectioning for microscopy ...........................................................................................