This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Phrathep, O-Phart Title: Biodiversity and physiology of Begonia iridoplasts General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. 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Biodiversity and physiology of Begonia iridoplasts O-Phart Phrathep A dissertation submitted to the University of Bristol in accordance with the requirements of the Degree of Philosophy in the Faculty of Life Sciences School of Biological Sciences, September 2019 Word count: 39,017 words I Abstract Iridoplasts and minichloroplasts are forms of modified plastid found in epidermal pavement cells of Begonia leaves. The repeated membrane system inside the iridoplasts has been shown to function as a multilayered photonic crystal generating iridescence while the special role of minichloroplasts remains unknown. The purposes of this study aimed to investigate the occurrence of iridoplasts, minichloroplasts and iridescence in phylogenetically diverse taxa of Begonia and the plastid locations in the leaf tissue as well as the plasticity of iridoplast and minichloroplast ultrastructure and iridescence under varying light intensities. Due to the new finding of the cryptic hypodermal iridoplasts, computational optical modelling method was introduced to determine their photonic properties. The results presented here confirm that both iridoplasts and minichloroplast exist in only dermal tissue of Begonia leaves, and they are both species- and dorsoventrally tissue- specific. Multilayered thylakoid stacked membranes within Iridoplasts function as photonic structure generating iridescence, resulting in reflected light in blue to green wavelengths in both real spectral measurement and predicted spectra by optical modelling. The changes of iridoplast and minichloroplast ultrastructure and iridescence under altered light conditions indicate their plasticity; however, they are not interconvertible. The finding of the existence of these modified epidermal plastids and their alterations responding to high and low light levels suggests the mechanisms underpinning adaptive strategies of plants to the changes of light environments. i Acknowledgement First and foremost, I would like to sincerely thank my supervisors, Dr Heather Whitney and Dr Jill Harrison, for their expert and patient supervision, encouragement, and support throughout my study making my PhD an incredible and unforgettable experience. I also thank the providers of my PhD scholarship, the Royal Thai Government and Mahidol Wittayanusorn School, Thailand, which allowed me to gain the excellent opportunity to study overseas and meet lots of fascinating people came worldwide in the UK. Secondly, I am indebted to Matthew Jacobs for being my first friend in Life Sciences and for his tireless help in both academy and life during my first two years. I would also like to express my gratitude to Dr Martin Lopez-Garcia and Dr Mark Hughes for their generous assistance and collaboration in optics expertise and Begonia phylogeny construction training, respectively. I am grateful for the patience and expertise of light and electron microscopy in the Wolfson bioimaging facility, particularly Judith Mantell, Gini Tilly, Tom Steward, Dr Chris Neal and Dr Sally Hobson. Manny thanks all botanic gardens meanthioned in this study for providing Begonia samples. I also thank all current and former members of the Whitney group; Nathan, Tom, Taryn, Kol, Cara, Hugh …, for their help, knowledge, company, making fantastic trips and conferences, and supplying good cake in every group meeting time. I am especially grateful to Alanna Kelly; Begonia expert, for her generous and tireless help as well as her remarkably horticultural expertise. Many thanks go to colleagues and friends in Plant lab 324, office 308 as well as other members across Life Sciences who I have been lucky enough to meet and collaborate. There are so many others, whom I could not mention here, but collectively their company and their encouragement made my PhD possible. Outside of the lab, my best friends – Bew, Aoy, Plasai, Jane, PPop, PKook, Pétanque and Mike-Possapon helped me unwind: many thanks, guys. I also thank Catherine and Dr Christopher Richards to make me feel like being home with mom and dad. Finally, I would like to thank my family for all their endless love and continued support and encouragement. ii Author’s declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ............................................................. DATE: .......................... iii List of contents Page Abstract I Acknowledgement II Author’s declaration III List of contents IV List of figures IX List of tables XIII Chapter 1 Introduction 1 1.1 Biological colour Begonia 1 1.1.1 Pigment-based colour 1 1.1.2 Structural-based colour 2 1.2 Structural colour 2 1.3 Structural colour in plants 3 1.3.1 Structural colour in reproductive structures: flowers and fruits 3 1.3.2 Structural colour in vegetative structures: leaves 3 1.4 Methods for observing the structural colour 4 1.5 Plastids 5 1.5.1 Proplastid 6 1.5.2 Etioplast 7 1.5.3 Chromoplast 7 1.5.4 Leucoplast (Amyloplasts, Elaioplast, and Proteinoplasts) 7 1.6 Chloroplast 8 1.6.1 Chloroplast structure 8 1.6.2 Chloroplast structural adaptation under low light 10 1.7 Modified chloroplast: Iridoplast and relatives 12 1.7.1 Iridoplast: modified chloroplast associated leaf iridescence 12 1.7.2 Diversity of iridoplasts and iridoplast-like structures in plants 15 1.7.3 Minichloroplast and other epidermal chloroplasts 16 1.7.4 Possible functions of iridoplasts 19 1.8 Begonia 19 iv Page 1.8.1 Begonia diversity 20 1.8.2 General characteristics of Begonia 20 1.8.3 Begonia leaf anatomy 22 1.9 Aims 23 Chapter 2 Occurrence of Begonia iridoplasts 24 2.1 Abstract 24 2.2 Brief Introduction 24 2.3 Aims 26 2.4 Materials and methods 26 2.4.1 Plant materials and growth conditions 26 2.4.2 Leaf imaging 35 2.4.3 Reflectance spectral measurement 35 2.4.4 Confocal laser scanning microscopy (CLSM) 36 2.4.5 Iridoplast ultrastructure study 36 2.4.6 Begonia phylogenetic tree construction 36 2.4.6.1 DNA extraction 39 2.4.6.2 PCR amplification 39 2.4.6.3 DNA sequencing 40 2.4.6.4 Phylogenetic analyses 40 2.4.7 Sampling methods and statistical analyses 40 2.5 Results 41 2.5.1 Leaf appearance 41 2.5.2 Microscopic observation of iridoplasts 45 2.5.3 Reflectance spectral measurement of iridoplast 48 2.5.4 Morphology and ultrastructure of iridoplasts and minichloroplasts 53 2.5.5 Molecular phylogeny of Begonia 57 2.6 Discussion 76 2.6.1 Leaf appearance 77 2.6.2 Microscopic observation of iridoplasts 78 2.6.3 Ultrastructure of iridoplasts and minichloroplasts 80 v Page 2.6.3.1 Locations in leaves and morphology of Begonia iridoplasts and 81 minichloroplasts 2.6.4 Association of iridoplast structure and Begonia leaf iridescence 82 2.6.5 Novel representative model of iridoplast 83 2.6.6 Possible functions of plastoglobuli in iridoplast 84 2.6.7 Microscopy techniques required for investigation of iridoplasts 84 2.6.8 Molecular phylogeny of Begonia and distribution of iridescence and iridoplasts 85 2.7 Conclusion 86 Chapter 3 Effect of light on Begonia iridoplast plasticity and iridescence 87 3.1 Abstract 87 3.2 Brief Introduction 87 3.3 Aims 90 3.4 Materials and methods 90 3.4.1 Plant materials and growth conditions 90 3.4.2 Growth analysis 93 3.4.3 Microscopic study of iridoplasts and iridescence 93 3.4.4 Statistical analysis 93 3.5 Results 94 3.5.1 Effect of light levels on Begonia growth 94 3.5.2 Effect of light levels on size of epidermal cells and size and quantity of 95 epidermal plastids 3.5.3 Effect of light levels on the ultrastructure of epidermal plastids 103 3.5.4 Effect of light levels on iridescence plasticity 119 3.6 Discussion 121 3.6.1 Effect of light intensity on Begonia growth and epidermal cell size 121 3.6.2 Effect of light intensity on size of iridoplast and minichloroplast 122