The impact of wind and mechanical stress on growth and development of Brachypodium distachyon stems Agnieszka Gladala-Kostarz A thesis submitted to Aberystwyth University for the degree of Doctor of Philosophy in Biological Sciences 18.09.2019 THESIS WORD COUNT 55326 DECLARATION This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed (Candidate) Date 18.09.2019 STATEMENT 1 This thesis is the result of my investigations, except where otherwise stated. Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed (Candidate) Date 18.09.2019 STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and inter-library loan, and for the title and summary to be made available to outside organisations. Signed (Candidate) Date 18.09.2019 ii “As many more individuals of each species are born than can possibly survive; and as, consequently, there is a frequently recurring struggle for existence, it follows that any being, if it vary however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life, will have a better chance of surviving, and thus be naturally selected. From the strong principle of inheritance, any selected variety will tend to propagate its new and modified form.” - Charles Darwin, On the Origin of Species, 1859 Introduction, page 5. iii ACKNOWLEDGEMENTS I would like to begin by expressing my deepest gratitude to my supervisors. To Dr Maurice Bosch for his patience, guidance, experience, knowledge and advice but also for huge empathy, support and understanding he offered during the last few years. Without his contribution, most of what has been achieved would not exist. To Prof. John Doonan for his guidance, help, valuable advice and confidence in my research. I am very privileged to have worked with them and extremely grateful to have been given the opportunity to work on this project. I also need to say thank you to Prof. Luis Mur for his help, advice and enthusiasm with metabolite analysis and Prof. Paul Knox for invaluable guidance with immuno and pectin experiments. Additionally, I would like to thank Alan Cookson for his assistance with SEM and to Dr Barbara Hauck and Dr David Walker for their help with cell wall composition analysis. I am also greatly indebted to Dr Candia Nibau, Dr Rakesh Bhatia, Emma Timms-Taravella, Dr Samantha Gill and Dr Fiona Cork for their help, assistance and technical advice from various biological sciences disciplines. A special acknowledgement I would like to address to Tom Thomas in the botany gardens for lovely morning chats during my glasshouse work, which brought the sun to even rainy weather. To everybody else who has helped me achieve my goals, I wish to express my deepest gratitude and ensure that any positive gesture will not be forgotten. This PhD would have been much harder without the help and support of my best friends. I am forever grateful to Joanna Wilinska for being always there, for her dedication, loyalty and for having a glass of wine when needed . I owe thanks to an extraordinary person in my life Martyna Andres-Brandyk I could say for everything, support, help, faith in me, dedication but especially for the most precious thing that can be given, for her time, it is priceless. I am honoured to have such wonderful people in my life. I do not know how to begin with saying thank you to the people who mean the world to me, my family, especially my mum Anna and dad Zbigniew, for their support, giving me the liberty to choose what I desired the most. I salute them both for the selfless love, care, pain and sacrifice they did to shape my life and for helping me to become iv the person I am now. I owe thanks to my brother for having that treasured feeling that I can always count on him no matter what. Also, I would like to express my thanks to my grandparents for their unconditional love and for enormous contribution in raising me in the most beautiful way I can imagine. Exceptional thanks to my grandma Jadzia for her support, faith in me and for valuable prayers. Special thanks go to my mother in law for her love and support. I would never be able to pay back the love and affection showed upon by all of you. Finally, I would like to dedicate this work to my soul mate, best friend and my dearest husband, Piotr. You believed in me in the way, which reached directly to my mind and heart. Thank you for encouraging me in all of my pursuits and inspiring me to follow my dreams. Thank you for your unfailing love, patience and for being so understanding to all of my strange life rules and for putting up with me through the toughest moments of my PhD. Love you forever. v SUMMARY The wind is an important abiotic stress from an agronomic and economic point of view. While the response of plants to various abiotic stresses is intensively studied, there is relatively little research on wind stress (WS) and mechanical stress (MS) in plants, especially in the grasses. This study aims to provide information on how wind stress and mechanical stress affect the growth and development of the model grass Brachypodium distachyon. In particular, the study focussed on the consequences of WS and MS on cell wall composition and architectural features of the stems, as well as phenotypic, molecular and metabolic responses. The study includes a comparison of two genotypes of Brachypodium, Bd21 and ABR6. Phenotypic observation demonstrated a reduction in main stem length and delayed flowering, reduction in seed yield and aboveground biomass for the two genotypes. More detailed analysis, including histology, anatomy, and composition analysis of stem cell walls, showed differences in response to WS and MS and between both genotypes Bd21 and ABR6. Investigation showed alterations in cell wall thickness of particular stem tissues as well as the organisation of stem tissues. Immunolocalisation using a range of monoclonal antibodies against non-cellulosic cell wall glycans, revealed differences in the labelling pattern obtained with pectin-related antibodies between treatments and genotypes. Mechanical stimulation enhanced pectin methylesterase activity and an increase in lignin content localised mostly in the cortex and interfascicular tissue. Differences in cell wall monosaccharide content were also observed. Sugar release after enzymatic hydrolysis was significantly reduced after both stress treatments. Furthermore, three-point-bending tests showed differences in the mechanical properties of stems exposed to WS/MS compared with control. In an attempt to provide functional information on the responses to WS and MS molecular and metabolomic analysis were performed. Molecular analysis revealed alterations in cell wall-related, LOX, and PME genes expression in response to WS and MS in both genotypes. Metabolic analysis unravelled pathways involved in response to mechanical stimulation. The study showed that wind and mechanical stress induce significant architectural changes across multiple scales, from the whole plant to organ, tissue, cellular and molecular level highlighting the complex nature of how plants respond to mechanical stimulation. Keywords: • Brachypodium distachyon • cell wall • wind stress • mechanical stress • mechanical stimulation • immuno-localisation • RT-PCR • metabolite profiling vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................... IV SUMMARY ......................................................................................................... VI TABLE OF TABLES ................................................................................................ XII TABLE OF FIGURES.............................................................................................. XIV ABBREVIATIONS ................................................................................................ XVII CHAPTER 1 : GENERAL INTRODUCTION ............................................................ 22 1.1. INTRODUCTION .................................................................................... 22 1.2. ABIOTIC STRESSES ................................................................................. 24 1.2.1. THIGMOMORPHOGENESIS .............................................................................. 24 1.2.1.1. Phenotypic responses.....................................................................................25 1.2.1.2. Anatomical and compositional responses......................................................28 1.2.1.3. Molecular responses ......................................................................................29 1.3. THE GRASS CELL WALL ............................................................................ 31 1.3.1. STRUCTURE OF THE GRASS CELL WALLS .............................................................. 31 1.3.1.1. Cellulose .........................................................................................................33 1.3.1.2. Hemicellulose .................................................................................................33 1.3.1.3. Lignin ..............................................................................................................36 1.3.1.4. Cell wall-bound hydroxycinnamic acids .........................................................37 1.3.1.5. Pectins ............................................................................................................38