Natural Genetic Variation in Arabidopsis Thaliana Photosynthesis
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Natural genetic variation in Arabidopsis thaliana photosynthesis Pádraic J. Flood Thesis committee Promotor Prof. Dr M. Koornneef Personal chair at the Laboratory of Genetics Director at the Max Planck Institute for Plant Breeding Research (MPIPZ) Cologne, Germany Co-promotors Dr M.G.M. Aarts Associate professor, Laboratory of Genetics Wageningen University Dr J. Harbinson Assistant professor, Horticulture and Product Physiology Wageningen University Other members Prof. Dr P.C. Struik, Wageningen University Prof. Dr J. de Meaux, University of Cologne, Germany Dr E. Murchie, University of Nottingham, United Kingdom Dr M. Malosetti, Wageningen University This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences (EPS). Natural genetic variation in Arabidopsis thaliana photosynthesis Pádraic J. Flood Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 30 October 2015 at 4 p.m. in the Aula. Pádraic J. Flood Natural genetic variation in Arabidopsis thaliana photosynthesis PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summary in English ISBN 978-94-6257-500-4 Contents Chapter 1 General Introduction 7 Chapter 2 Natural genetic variation in plant photosynthesis 21 Chapter 3 Phenomics for photosynthesis, growth and reflectance in Arabidopsis thaliana reveals circadian and long-term fluctuations in heritability 43 Chapter 4 QTL analysis through time of Arabidopsis thaliana for photosynthesis, growth and chlorophyll reflectance reveals the dynamic nature of complex trait architecture 75 Chapter 5 Genome wide association mapping for photosynthesis in Arabidopsis thaliana 117 Chapter 6 Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana – is it caused by genetic variation in the STN kinases? 149 Chapter 7 Construction and preliminary analysis of a nucleotype-plasmotype diallel in Arabidopsis thaliana 175 Chapter 8 Whole genome hitchhiking on an organelle mutation 195 Chapter 9 General Discussion 211 References 231 Summary 257 Acknowledgements 261 About the Author 269 Chapter 1 General Introduction -On writing a PhD thesis I think perhaps the most important problem is that we are trying to understand the fundamental workings of the universe via a language devised for telling one another when the best fruit is. -- (Terry Pratchett) General Introduction Where light becomes life It is a little recognized fact that plants grow upside down. Yes, you read that right, I did just state that plants grow upside down. Before you go and throw this thesis in the rubbish bin, bear with me while I reveal the logic behind this apparent absurdity. The first step towards elucidating the curious upside down nature of plant growth was taken by the Flemish chemist and physiologist Jan Baptist van Helmont. In 1643 he planted a willow tree weighing 5 pounds (2.2 kg) in a pot containing 200 pounds (90 kg) of soil previously dried in a furnace. For the next five years he watered this pot with either rainwater or distilled water after which he removed the willow and found that it weighed 169 pounds and 3 ounces (77 kg) whilst the soil had only lost 2 ounces (57 grams). From this he erroneously concluded that the tree obtained its mass from the water. It took about 150 years and the endeavors of many scientists, in which Joseph Priestley, Jan Ingen-Housz, Jean Senebier and Nicholas- Théodore de Saussure played prominent parts, to come to the correct conclusion that air, specifically carbon dioxide (CO2), is responsible for most of the solid mass of plants (Hill, 2012). Arising from this, the key insight into the upside down nature of plants was garnered: plants are largely composed of air not soil, in this respect they are coalesced air which grows into the earth and not from it. This particular realization was one of the main motivations for me to pursue a PhD on the process which makes this remarkable phenomenon possible: photosynthesis. Photosynthesis is the fundament upon which virtually all life on Earth depends1. It traps the light of the Sun in chemical bonds providing the energy necessary for life to maintain its improbable entropic state. In the case of oxygenic photosynthesis it also produces oxygen as a waste product which, via oxygenic respiration, can be used to extract much more of this stored sunlight than would otherwise be possible. Thus, with spectacular providence, oxygenic photosynthesis both injects more energy and matter into the biosphere and provides the necessary chemical tools for efficiently recovering this energy when needed. Oxygenic photosynthesis is an ancient biological innovation dating back to over 3 billion years ago (Hohmann-Marriott and Blankenship, 2011); its evolution was a pivotal event in the history of life as it changed the chemistry of the planet from partially reducing to 1 There are some communities of organisms which live on hydrothermal vents and are energetically independent of photosynthesis, however without the generation of the protective ozone layer which oxygenic photosynthesis gave rise to, it is doubtful there would be much water left even to sustain this life. 8 | P a g e Chapter 1 oxidizing. As already mentioned, it resulted in a massive increase in the amount of energy and matter flowing into the biosphere2. It also facilitated the development of the ozone layer which has protected the Earth from much of the ultraviolet radiation emitted by the Sun. In summary, oxygenic photosynthesis has been absolutely key in the development of a suitable environment on Earth within which life can thrive. Finally, unlike many key evolutionary innovations, such as multicellularity, eyes and intelligence, oxygenic photosynthesis only evolved once. The singularity of this event is striking and as such it is widely considered a key event in the history of life3. The unlikely nature of oxygenic photosynthesis is perhaps best explained by its sheer complexity; it is thought to be the most complex energy transducing process in biology (Shi et al., 2005). In plants this complexity is taken a step further by the separation of genes coding for photosynthetic proteins into two discrete genetic lineages, the nuclear genome and the chloroplast genome, or plastome. Both genomes encode subunits of large protein complexes, such as photosystem II (PSII), which must be expressed and manufactured in tight coordination and then fit together in a precise arrangement. One mistake can be fatal as photosynthetic energy transduction deals with high energy processes that generate reactive intermediates, which can, in particular, react with molecular oxygen. Small errors in the assembly and operation of these systems can lead to the creation of free radicals such as superoxide, which in turn can cause widespread and indiscriminate cellular damage. This high level of complexity and interdependence is thought to have constrained the evolution of photosynthesis by limiting the number of options upon which evolution can work (Shi et al., 2005). The observation that core photosynthetic proteins are highly conserved, combined with little evidence that crops were limited by their photosynthetic capacity (usually referred to as source limitation) has led to a general lack of enthusiasm for improving photosynthesis. Since the turn of the millennium this has begun to change with numerous researchers thinking of ways to improve photosynthesis (Richards, 2000; Long et al., 2006). In general the improvement approaches touted have taken an engineering angle (Bar-Even et al., 2010; von Caemmerer and Evans, 2010; Raines, 2011; Furbank et al., 2015; Long et al., 2015; Ort et al., 2015) with little attention paid to the potential of natural genetic variation as a source for improving 2 In this context when I say biosphere I am referring to the sum total of all living things on the planet not the specific region they inhabit, confusingly the term is used somewhat interchangeably and I could not find a more precise word. 3 The only other evolutionary step which is also thought to be extremely unlikely is the evolution of eukaryotes, this also happened only once and has been hypothesized to be a key requirement for complex multicellular life to evolve (Lane and Martin, 2010). 9 | P a g e General Introduction photosynthesis (Flood et al., 2011; Lawson et al., 2012). In this thesis I aim to address this lack of attention by surveying natural genetic variation in photosynthesis in the model plant species Arabidopsis thaliana, hereafter referred to as Arabidopsis. Arabidopsis is an unassuming plant, most people have probably either stepped on it, sprayed it with herbicide or pulled it from their flower bed without ever really seeing it. It is small, forms a rosette of leaves from which a thin wispy inflorescence grows and gives rise to a host of tiny, odorless white flowers. To the untrained eye it is perhaps the epitome of unimpressive. But beneath this banal exterior Arabidopsis holds a beguiling secret: it has become our gateway into the wondrous world of plants. Arabidopsis is the most unique crop there is; its fruit is the fruit of knowledge. Over the past half century this unassuming weed has been used to probe the inner workings of plants. In the year 2000 it became the first plant ever to have its whole genome sequenced (The Arabidopsis Genome Initiative, 2000) and it continues to be at the forefront of fundamental plant science research (Koornneef and Meinke, 2010). Recently, there has been a growing interest in investigating natural genetic variation in Arabidopsis as such variation is often evolutionarily and ecologically informative (Koornneef et al., 2004; Alonso-Blanco et al., 2009). Prior to this recent interest in natural variation, most studies into Arabidopsis gene function worked with genetic variation created in the laboratory.