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The effect of UV-B radiation on flavonoid accumulation and gene expression in Marchantia polymorpha A Thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biochemistry at Lincoln University by William Armstrong Clayton Lincoln University 2017 Abstract of a Thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biochemistry. Abstract The effect of UV-B radiation on flavonoid accumulation and gene expression in Marchantia polymorpha by William Armstrong Clayton Marchantia polymorpha is the closest living representation of the earliest land plants and as such may give insight into the response that allow the first land plants to transition from the aquatic environment onto the terrestrial. One such adaption allowing this transition was to that of UV-B radiation and is the main focus of this study.This thesis investigates the effect of UV-B radiation on the basic land plant M. polymorpha and its responce through the production of secondary metabolites, in particular flavonoids. How flavonoid compounds may protect M. polymorpha and the gene regulation that contributes towards stress related defense and secondary compound production is also investigated. M. polymorpha plants were subject to UV-B fluence at two seperate levels to stimulate both an acclimation and stress related response. Plants were then measured for the production of UV-B absorbing compounds, in particular the flavonoids, and also the localisation in thallus tissue of such compounds. The genetic response was also analysed using a combination of RNA-seq and nCounter techniques. Specific mutant plants with altered flavonoid amounts were also analysed to help further determine the genetic interactions that contribute to protection. M. polymorpha was found to respond to UV-B through enhanced levels of UV-B absorbing compounds and in particular flavonoids, of which the flavones were the most predominant. Apigenin-based flavones were found in the highest amounts followed by luteolin-based ii flavones. The ratio of Apigenin to Luteolin-based flavones was seen to shift towards luteolin in high UV-B responses. Apigenin was suggested to be required for UV-B screening functions under low fluence while high fluence treatments, that induce ROS may require Luteolin- based flavones that are able to scavenge ROS more effectively. Under low fluence conditions flavone compounds accumulated strongly in the epidermal layers suggesting a strong UV-B screening function. RNA-seq revealed that M. polymorpha responds to UV-B through the UVR8 pathway similar to that of higher plants. We determined the UVR8 pathway in M. polymorpha contains the main regulators COP1, HY5 and RUP1 and act to enhance flavonoid biosynthetic genes such as CHS, CHI and FNS. RNA-seq and nCounter analysis also revealed differential expression in stress related genes that may offer protection independently of the UVR8 pathway and flavonoids. Mutant lines with altered flavonoid amounts were also tested under UV-B irradiance and showed that lower flavones concentrations resulted in enhanced damage to plant thallus. Elevated flavones resulted in enhanced protection and a reduction in thallus damage and loss of flavones altogether resulted in severe stunting in plants grown under UV-B. Overall we determined that M. polymorpha responds through UVR8 for the production of flavone compounds and this production results in protection against the deleterous effects of UV-B. Such adaptive mechanisms likely assisted early land plants to transition from aquatic to land environments by reducing UV-B burden. iii Acknowledgements I first would like to thank my supervisors, Professor Brian Jordan, Dr Kevin Davies, Dr Kathy Schwinn and Dr Simon Deroles. Without your knowledge and support none of this would have been possible. Special thanks must go to Dr Nick Albert who worked tirelessly in creating the transgenics and taught me so much. It was truly great to have someone of Nick’s talent to learn off and support me through my experiments. Thank you. The support of my two main supervisors, Brian Jordan and Kevin Davies, deserve special mention as without their support and valuable knowledge none of this would have been possible. Thanks to my lab mates at Lincoln for helping out when needed. Thank you to Scott for showing me the ropes around the lab and helping me with equipment and advice. Also thank you to Darrell for managing the lab and always being able to help out when needed. Thanks to Jenny Zhao and Rosy Tung for HPLC expertise and letting me use the plate reader. Also thanks to Steven Stillwell for his help with setting up the UV cabinets and providing me with the precious cellulose acetate in order to keep the experiments going. Thanks must go to Plant and Food research for providing me with the opportunity to study and also to the Marsden fund for funding the project. A special thanks to my family and friends for supporting me through my study. Not just in my PhD but throughout all my University endeavours. Your support and encouragement was truly invaluable and kept me going even in the toughest times. iv Table of Contents Contents Abstract .................................................................................................................................................... ii Acknowledgements ................................................................................................................................. iv List of Tables ............................................................................................................................................ ix List of Figures ........................................................................................................................................... x Abbreviations ........................................................................................................................................ xvi Chapter 1: Introduction ........................................................................................................................... 1 1.1 Evolution of land plants..................................................................................................................... 1 1.2 Adaption to stress on land ................................................................................................................ 2 1.2.1 Desiccation tolerance ................................................................................................................. 2 1.2.2 Thermal tolerance ...................................................................................................................... 3 1.2.3 Adaption to UV ............................................................................................................................... 4 1.3 UV induced damage in plants ........................................................................................................... 5 1.3.1 DNA damage............................................................................................................................... 5 1.3.2 Damage to photosynthesis......................................................................................................... 6 1.3.3 Reactive Oxygen Species ............................................................................................................ 7 1.4 UV response in plants ........................................................................................................................ 9 1.4.1 Specific UVR8 mediated UV-B response pathway in plants ....................................................... 9 1.4.2 Non-Specific UV-B responses ................................................................................................... 13 1.5 Photoperception of Non-UV-B light ................................................................................................ 14 1.5.1 Cryptochrome .......................................................................................................................... 15 1.5.2 Phototropins............................................................................................................................. 16 1.5.3 Phytochrome ............................................................................................................................ 17 1.6 Plant pigments ................................................................................................................................ 18 1.6.1 Chlorophylls .............................................................................................................................. 18 1.6.2 Carotenoids .............................................................................................................................. 20 1.6.3 Betalains ................................................................................................................................... 22 1.6.4 Proanthocyanidins .................................................................................................................... 22 1.7 Flavonoids ....................................................................................................................................... 23 1.7.1 Chalcones ................................................................................................................................. 24 1.7.2 Flavones and Flavonols ............................................................................................................ 24 1.7.2.1 Function of Flavones ......................................................................................................... 25 1.7.2.2 Function of the Flavonols .................................................................................................. 26 1.7.3 Anthocyanins ............................................................................................................................ 27 1.7.4 3-Deoxyanthocyanins ............................................................................................................... 29 v 1.8 Pigments of Green Algae ................................................................................................................. 32 1.9 Liverworts ........................................................................................................................................ 34 1.9.1 Physiology of Liverworts ........................................................................................................... 34 1.9.2 Liverworts life cycle ................................................................................................................. 39 1.9.3 Flavonoid pathway in Marchantia polymorpha ....................................................................... 40 Chapter 2: Materials and Methods ....................................................................................................... 43 2.1 Controlled Environment Experiments ............................................................................................. 43 2.1.1 Growth Conditions.................................................................................................................... 43 2.1.2 UV environment ....................................................................................................................... 44 2.2 Biochemical Analysis........................................................................................................................ 45 2.2.1 Methanol extraction ................................................................................................................. 45 2.2.2 Total UV absorbing Compounds: .............................................................................................. 46 2.2.3 Ultra High Performance Liquid Chromatography (UHPLC) ....................................................... 46 2.2.4 Total RNA isolation ................................................................................................................... 47 2.2.5 DNase treatment ...................................................................................................................... 48 2.2.6 Total RNA quantification .......................................................................................................... 48 2.2.7 cDNA synthesis ......................................................................................................................... 49 2.2.8 Quantitative Real Time PCR (qRT-PCR) ..................................................................................... 49 2.2.9 Bioanaylser ............................................................................................................................... 50 2.2.10 Reactive Oxygen Species Fluorescent Reporter ..................................................................... 50 2.2.11 Chlorophyll quantification ...................................................................................................... 51 2.2.12 Dissecting microscopy ............................................................................................................ 51 2.2.13 Fluorescent microscopy for flavonoid visualisation ............................................................... 51 2.2.14 RNA sequencing ...................................................................................................................... 52 2.2.15 nCounter Analysis ................................................................................................................... 52 2.2.16 Statistics .................................................................................................................................. 53 Chapter 3: The physiological response of M. polymorpha to UV-B irradiance ..................................... 54 3.1 Introduction ................................................................................................................................. 54 3.2 Results ......................................................................................................................................... 55 3.2.1 M. polymorpha response to high and low UV-B fluence .......................................................... 55 3.3 Discussion .................................................................................................................................... 61 Chapter 4 ............................................................................................................................................... 65 4.1 Introduction ................................................................................................................................. 65 4.2 Results ......................................................................................................................................... 66 4.2.1 M. polymorpha production of UV-B absorbing compounds .................................................... 66 vi 4.2.2 Flavones contribute to the change in UV-B absorbing compounds ......................................... 67 4.3 Flavonoid localisation in M. polymorpha under UV-B conditions ............................................... 73 4.4 Discussion .................................................................................................................................... 77 Chapter 5: The genetic response of M. polymorpha to UV-B ............................................................... 83 5.1 Introduction................................................................................................................................. 83 5.2 Results ......................................................................................................................................... 84 5.2.1 RNA sequencing of M. polymorpha under UV-B conditions ................................................ 84 5.2.2 Fold change regulation of putative gene families in M. polymorpha under UV-B conditions ....................................................................................................................................................... 95 5.2.3 Flavonoid regulator genes and biosynthetic gene changes in M. polymorpha under UV-B conditions .................................................................................................................................... 103 5.3 Discussion .................................................................................................................................. 109 5.3.1 RNA sequencing of M. polymorpha under UV-B conditions .............................................. 109 5.3.2 Fold change regulation of putative gene families in M. polymorpha under UV-B conditions ..................................................................................................................................................... 114 5.3.4 Flavonoid regulator genes and biosynthetic gene changes in M. polymorpha under UV-B conditions .................................................................................................................................... 118 5.3.4 Summary ............................................................................................................................ 121 Chapter 6: Mutation analysis of flavonoid response genes ................................................................ 123 6.1 Introduction............................................................................................................................... 123 6.2 Results ....................................................................................................................................... 124 6.3 Discussion .................................................................................................................................. 140 6.3.1 UV-B response mutants of M. polymorpha have significant changes in flavone production ..................................................................................................................................................... 140 Chapter 7: nCounter analysis of M. polymorpha genes under low fluence UV-B conditions ............ 147 7.1: Introduction ............................................................................................................................. 147 7.2 Results ....................................................................................................................................... 147 7.2.1 Analysis of UVR8 pathway expression under UV-B conditions .......................................... 147 7.2.2 Analysis of putative flavonoid transcription factors .......................................................... 152 7.2.3 Analysis of flavonoid biosynthetic genes ........................................................................... 158 7.2.4 Analysis of putative stress and indirect pathway genes in response to UV-B ................... 166 7.3 Discussion .................................................................................................................................. 181 7.3.1 Analysis of UVR8 pathway expression under UV-B conditions .......................................... 182 7.3.2 Analysis of putative flavonoid transcription factors .......................................................... 183 7.3.3 Analysis of flavonoid biosynthetic genes ........................................................................... 184 7.3.4 Analysis of putative stress and indirect pathway genes in response to UV-B ................... 187 vii Chapter 8: General discussion ............................................................................................................. 192 References ........................................................................................................................................... 198 Appendices .......................................................................................................................................... 223 viii List of Tables Page Table 5.1 The 20 most up regulated and down regulated genes in M. polymorpha under 85 high fluence UV-B treatment at 4 hours. Table 5.2 The 20 most up regulated and down regulated genes in M. polymorpha under 87 high fluence UV-B treatment at 1 day Table 5.3 The 20 most up regulated and down regulated genes in M. polymorpha under 88 low fluence UV-B treatment at 1 day Table 5.4 Differentially expressed genes of M. polymorpha under UV-B treatment 107 ix