Comparative study of cell walls during wheat and rice grain development By Richard Andrew Palmer Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences September 2015 1 The candidate confirms that the work submitted is his/her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. In chapter 5, the cloning of monoclonal antibody-secreting cell lines and the antibody characterisation was performed by Sue Marcus. Preparation of the AGP wheat extract for immunisation was performed by Dr Alison Lovegrove. In chapter 5, Figure 5.3 was produced by Sue Marcus. The experiments and sample preparation leading to this image were performed by her. In chapter 5 all production of the RNAi lines was performed by Dr Mark Wilkinson. In chapter 5, high pressure freezing of null and RNAi wheat lines waas conducted by Dr Paola Tosi. All paraformaldehyde fixation, all sectioning and immunohistochemistry were performed by the candidate. 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. The right of Richard Andrew Palmer to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. © 2015 The University of Leeds and Richard Andrew Palmer 2 ACKNOWLEDGEMENTS I would like to thank my supervisors, Paola Tosi; Peter Shewry and Paul Knox for their continuous support, incisive comments and understanding during the past four years of this PhD. I greatly appreaciate all your time and efforts to get me through this in one piece! I would also like to thank Fabienne Guillon at INRA Nante for her generosity in providing me with several of their cell wall antibodies, which have helped my to complete this project. 3 ABSTRACT The cell wall polysaccharides of wheat and rice endosperm represent an important source of dietary fibre with up to 50% of the dietary fibre intake in western diets deriving from cereal consumption. Plant cell walls are complex structures composed of many interlinking polysaccharide chains as well as lignin, phenolics and some proteins. The significance of many of these molecules has yet to be elucidated, however cell walls have been shown to be rapidly modified during growth and differentiation demonstrating that the cell wall is a dynamic structure modified in muro to adapt to changing biological constraints. Wheat and rice present near synchronous developmental cycles and significantly different endosperm cell wall compositions, allowing the localization of these polysaccharides to be related to developmental changes. Monosaccharide analysis has been widely used on mature endosperm and flour sample in cereal grains to provide an overview of cell wall composition, but no previous studies have considered different developmental stages. In both wheat and rice four distinct phases of cell wall deposition were detected, with 4-8 days after anthesis (DAA) and 12-20 DAA showing the greatest levels of depositions in all monosaccharides. After 20 DAA significant deposition of pectic polysaccharides was detected in both species, which may reflect preparations for grain dehydration. Monoclonal antibodies specific to cell wall polysaccharides and immunofluorescence microscopy were used to determine the spatial and temporal locations of these polysaccharides. A conserved sequence of polysaccharide deposition during cellularisation was also seen in both species, matching that reported in barley grains. Arabinogalactan-petides (AGPs) are a significant component of wheat and rice grains and through the use of novel wheat AGP monoclonal antibodies they were shown to be localised either at the plasma membrane or in the cytoplasm, contrary to previous hypotheses that they may be cell wall proteins. 4 TABLE OF CONTENTS Chapter 1: Introduction ............................................................................................ 22 1.1 General introduction ...................................................................................................... 22 1.2 Seed development .......................................................................................................... 22 1.2.1 Pollination and Fertilization .............................................................................................. 22 1.2.2 Endosperm development .................................................................................................. 23 1.2.3 Embryo development........................................................................................................... 25 1.3 Differences in wheat and rice transport pathways ........................................... 25 1.4 Seed protein ...................................................................................................................... 27 1.4.1 Storage proteins ..................................................................................................................... 27 1.5 Starch ................................................................................................................................... 31 1.5.1 Starch composition ............................................................................................................... 31 1.6 Cell wall components .................................................................................................... 33 1.6.1 Arabinoxylan (AX) ................................................................................................................. 34 1.6.2 Mixed-linkage β Glucan (MLG) ....................................................................................... 37 1.6.3 Heteromannans ...................................................................................................................... 39 1.6.4 Callose ........................................................................................................................................ 40 1.6.5 Pectic polysaccharides ....................................................................................................... 40 1.6.5.1 Homogalacturonan ......................................................................................................................... 41 1.6.5.2 Rhamnogalacturonan-I (RG-I) .................................................................................................. 42 1.6.5.3 RG-II ....................................................................................................................................................... 45 1.6.5.4 Biosynthesis of pectin ................................................................................................................... 46 1.6.6 Cellulose .................................................................................................................................... 48 1.6.6.1 Cellulose biosynthesis .................................................................................................................. 49 1.6.6.2 Function ................................................................................................................................................ 49 1.6.7 Lignin ........................................................................................................................................... 50 1.6.8 Arabinogalactan peptides .................................................................................................. 51 1.7 Gradients in grain composition. ............................................................................... 52 1.8 Usage and limitations of monoclonal antibodies .............................................. 53 1.9 Aims of project ................................................................................................................. 53 Chapter 2: Materials and Methods ........................................................................ 56 2.1 Plant materials .................................................................................................................. 56 2.2 Immunolocalization of cell wall polysaccharides wheat and rice grains. ........................................................................................................................................................ 57 2.2.1 Light microscopy and immunofluorescence analysis .......................................... 57 5 2.2.2 Sample preparation for high pressure freezing. .................................................... 57 2.2.3 Indirect immunofluorescence labelling and histochemical staining for light microscopy. .......................................................................................................................................... 58 2.2.3.1 Enzymatic unmasking................................................................................................................... 60 2.3 Histochemical stains ..................................................................................................... 60 2.3.1 General morphology ............................................................................................................ 60 2.3.2 Protein body staining ..........................................................................................................