Theses - Plant Sciences

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Exploring strategies to enhance pollination and yield in the field bean, Vicia faba L.
Moscrop, Jake
Vicia faba is a legume crop valued for its high protein content and ability to fix nitrogen. The UK is a key producer of faba beans, which are likely to be increasingly important as a less environmentally harmful protein source. However, without insect pollination, V. faba yield declines by 32.9% on average. When faced with continuing bee declines, it is imperative that more is done to understand how floral traits of V. faba may be used to improve crop yield through pollinator attraction. In this project, novel variation has been identified between commercial lines of V. faba in reward, attraction, and access traits. Using this variation, a hypothesis was formulated that V. faba lines possessing floral traits considered to be more attractive to bees would receive more bee visits. Data from two years of field trials supported this hypothesis, with bees showing preference for lines with more flowers and superior nectar sugar content. Field trials also supported the hypotheses that open pollination would have a positive effect on V. faba yield, and that lines with more attractive floral traits would receive a larger yield benefit with open pollination. These results have shown for the first time that V. faba floral trait variation affects bee attraction in the field and has significant effects on the pollination of the crop. Following field trials, preferences experiments using Bombus terrestris foragers in controlled condition were used to identify V. faba floral traits likely to be most important for bee attraction. Together, field and controlled condition experiments suggested that floral colour, nectar concentration, display size, and scent are traits most likely to increase pollinator attraction to the crop. These experiments have also contributed to the wider understanding of pollinator attraction, adding to evidence that bees show preference for purple colours. The evidence presented in this thesis suggests that by growing existing Vicia faba lines with floral traits including purple flowers with more concentrated nectar, farmers can ensure better crop yield in environments where pollinators are present. In addition, floral traits should be considered as breeding targets by V. faba breeders, as they have great potential to increase crop pollination and support wild pollinator populations.
Open Access
The effects of novel genetic diversity on yield and yield traits in a hybrid wheat pre-breeding programme
Argirou, Yeorgia
Hybrid breeding, where two inbred lines are crossed to create superior F1 plants, has the potential to increase yield and yield stability in wheat. However, commercialisation of hybrid wheat is limited by high production costs but could be offset by increasing the yield. Introducing new diversity into hybrid breeding programmes could increase yield through improved traits and by increasing the genetic distance between hybrid parents which is hypothesised to increase heterosis. In this project, synthetic hexaploid-derived wheat (SHW) and tetraploid x hexaploid wheat (THW) pre-breeding lines containing novel diversity were used to assess their potential for hybrid breeding. Hybrid wheat is sown at a lower density than conventional line-bred wheat and so traits that increase yield at lower sowing densities may be valuable in a hybrid context. Thirty ‘large eared’ SHW and THW lines were sown at typical line-bred (100%) and hybrid (70%) sowing densities in a multi-year field trial to see if the large ear trait resulted in yield advantages at the lower density. Most ear and seed yield component traits were higher at the hybrid sowing density, with yields maintained or only slightly reduced at the hybrid density. However, this was mostly influenced by tiller number and ear traits often showed a trade-off with tiller number. In a second trial series, 99 hybrids were created and tested in five field trials across UK, France, and Germany over two years to test whether increased genetic diversity could lead to improved hybrid performance. Hybrids were developed by crossing diverse and commercial elite male parents with three male-sterile testers as female parents. Results were variable with some pre-breeding hybrids performing well and hybrids generally showing better yield stability than inbreds. A separate trial investigated the optimal sowing density for yield in hybrid wheat. Across five different hybrids, the optimal sowing density ranged from 80-100% relative to inbreds, which is above the typical hybrid sowing rate of 70%. Crosses were also made between different pre-breeding lines used as males in the hybrid trials to create new material with recombined diversity and to reduce their relatedness to their backcrossed parent, Robigus. Lines from 15 crosses have been taken through to the F6 plant stage after being selected for beneficial traits and will be used in forward breeding. A transcriptomics study was carried out to investigate how gene expression differed between two parents and their reciprocal F1 hybrids. Two parents contrasting for yellow-rust (YR) resistance and their reciprocal hybrids were inoculated with YR to look at their transcriptional differences at 0, 3, and 5 days post inoculation (DPI). Large numbers of differentially expressed genes were found between all four genotypes with the hybrids mostly showing more similar expression patterns to their YR-susceptible parent, whilst the YR resistance appeared to be dominant. 95.3-99.9% of genes exhibited additive expression and the genes that exhibited non-additive expression were mostly overdominant at 0 and 3 DPI, and partially dominant at 5 DPI. In conclusion, these findings illustrate the complexity of hybrid breeding in hexaploid wheat. They show that diverse material does show potential for hybrid improvement; however, targeting single traits such as the large-eared character in isolation will probably be insufficient to drive sustained improvements in yield and yield stability.
Open Access
Genetic control and prediction of milling and baking quality for UK wheat breeding
Fradgley, Nicholas
Bread wheat for human consumption makes a great contribution to health and nutrition of the growing global population, but competition for human digestible grains that are fed to livestock has concerning implications for sustainability and food security. Specific quality requirements must be met for wheat crops to be suitable for bread making and these are both genetically and environmentally controlled. In the UK, breeders have historically focussed more on increasing yield rather than quality due to the difficulty in selecting for multiple low throughput and high cost quality traits. This thesis aims to redress this imbalance by taking a quantitative genetics approach to enable enhanced selection for milling and baking quality in collaboration with the DSV UK breeding programme. A wheat multi-parent advanced generation intercross population was used to investigate the genetic control of multiple wheat quality and micronutrient traits. This analysis identified multiple quantitative trait loci (QTL) with co-locating pleiotropic effects that could explain much of the complementary and antagonistic relationships among these traits. Of note, a QTL that co-located with the awn length inhibitor locus on chromosome 5A was found to consistently increase grain calcium content while not decreasing grain specific weight, despite the established negative correlation between these two traits. Principal component based multi-trait analysis increased the power to detect novel QTL that have effects that contradict overall trait correlations so may be useful to optimise antagonistic trait trade-offs. Genomic prediction of quality and loaf baking quality traits were then investigated in a panel of released high quality wheat varieties and recent breeding lines. Historical trends in these traits and changes in frequency of QTL alleles identified through genome wide association analysis revealed evidence for breeders’ selection for decreased protein content but increased loaf baking quality for the Chorley Wood Baking Process. QTL identified here have direct application for improvement of quality traits, such as grain specific weight and Hagberg falling number, for which little improvement has been made in recent decades of breeding. However, most QTL identified here could only explain a small proportion of the heritability of most complex quality traits. Genomic selection was shown to be highly applicable for prediction of costly loaf baking quality traits and offer increased prediction accuracy at reduced costs in comparison to phenotypic selection based on early-stage predictive traits. Stability of wheat quality traits across environmental variation is also an important target for selection. A large dataset of long-term field trials in the UK was analysed with genetic marker and pedigree data to characterise genotypes and weather and soil covariates to characterise environments. Cross validation of untested genotypes in untested years demonstrated that prediction models were able to successfully predict environmental and genotype by environment interaction effects. Predictions into future environments simulated from climate projection models enabled prediction of climate change impacts on UK wheat quality and the potential for breeding to mitigate these impacts. This project provides several quantitative genetics tools and resources for enhanced selection of wheat milling and baking quality with direct relevance to a UK breeding programme.
Open Access
Tulipa: the taxonomy and evolutionary history of the genus and its impact on conservation priorities in Central Asia
Wilson, Brett
Tulips are one of the most recognisable plants with their current horticultural trade estimated as a billion-euro industry. This trade initially relied heavily on wild specimens but now relies less on natural diversity. Yet, wild tulips are an important genetic reserve for future breeding efforts, especially for disease resistance. They also have significant cultural value, can act as ecological indicators, and support insect populations. Their taxonomy is notoriously complex, complicating their study and conservation. Most taxa grow in Central Asia, which has been proposed as the place of origin of this genus, albeit with limited evidence. Many tulips are declining in this area, but there is no cohesive regional overview of the genus. Here, we address phylogeny and taxonomy within the genus, then using this insight to explore the evolutionary history of Tulipa and its potential to inform conservation priorities in Central Asia. First, we used modern phylogenetic techniques, with extensive sampling of the genus including large amounts of wild material collected during three fieldwork seasons, to generate both a plastome based and 35S rDNA phylogeny. These phylogenies allowed us to make a number of taxonomic decisions with respect to the synonymization and reinstatement of a number of species. We also reorganised the higher-level taxonomic groups of this genus recognising a new subgenus, Eduardoregelia, and simplifying the sections of this genus, primarily merging Tulipa, Tulipanum, Lanatae, Vinistriatae, and Spiranthera into one broader section. Within this work we identified a new species, Tulipa toktogulica, which we formally describe within this thesis as well. Second, we used molecular dating techniques to estimate the ages of nodes on the tulip species phylogeny. With this dated phylogeny we modelled the biogeographical history of the genus, generated a lineage through time plot, and assessed the phylogenetic signal for the trait of genome size, which has commonly been used in the taxonomy of tulips including in the description of new species. We then assimilated dates, biogeography, and geological history to propose how this genus diversified and migrated to its current distribution. We confirmed a broader Central Asian origin of this genus, highlighting the importance of this region for the diversification of this genus throughout its evolutionary history, and linking speciation to aridification, mountain building, and global cooling. Finally, we modelled the impacts of climate change on tulip species in Central Asia showing the large negative impact this threat will have. This information was then used in the Red Listing of a range of Central Asian tulip species, which was undertaken at a workshop in Bishkek in Spring 2022. Through this process, a large amount of information was collated and many taxa from this region were assigned a threat status. Using data from the Red List assessments we undertook several post-analyses, showing that national assessments often overinflate threat status, as well as calculating EDGE scores in order to stimulate evolutionarily informed conservation efforts. Overall, we have provided a foundation for the development of a regional tulip conservation strategy and improved conservation prioritisation, both of which directly support the work of our iCASE partner Fauna & Flora International.
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Vitamin B12-mediated physiology, mutualisms, and gene regulation studies in Chlamydomonas reinhardtii and Phaeodactylum tricornutum
Newsad, Shelby
Many organisms engage in symbiotic relationships that govern, to varying degrees, the function of ecosystems and their evolutionary history. It is important to understand the origins and dynamics of these systems. Especially important is the aquatic ecosystem, which is essential for approximately 50% of global primary production, and where many photosynthetic algae participate in symbiosis with bacteria where the latter provide vitamins such as cobalamin (vitamin B12) in exchange for fixed carbon from the algae. Vitamin B12 is used as a cofactor by methionine synthase (METH), but many algae such as the freshwater chlorophyte, Chlamydomonas reinhardtii, and the marine diatom, Phaeodactylum tricornutum, also have a B12-independent form of methionine synthase (METE). This means that they can utilize METH if B12 is available, but when it is absent or in limited supply, they use METE. To study the influence of vitamin exchange, CRISPR (clustered regularly interspaced short palindromic repeats) was used to make gene-edited metE mutants of both species. This enabled the study of the physiology of newly B12 dependent algae and mutualisms with B12-producing bacteria. As well as the role of B12 as a cofactor, it also acts to repress gene expression. Most notably, METE shows rapid and robust repression in the presence of B12 in both C. reinhardtii and P. tricornutum. This has been previously shown to occur through the promoter regions in both organisms. This dissertation has three main parts which are i) physiological and genetic consequences of acquired vitamin auxtrophy in C. reinhardtii and P. tricornutum; ii) bacterial mutualisms underpinned with B12 in C. reinhardtii and P. tricornutum; and iii) a comparative study of the regulation of C. reinhardtii and P. tricornutum METE using fluorescent reporters. To understand the physiological consequences of vitamin auxotrophy, the B12 requirement and response characteristics were determined for growth, growth rate, cell size changes, and transcript response in vitamin deplete and replete conditions. The B12-dependent C. reinhardtii had a higher vitamin requirement than P. tricornutum. When looking at cell size, C. reinhardtii was shown to drastically increase size in vitamin deprivation, which did not occur with P. tricornutum. One-carbon (C1) metabolism, which is important for amino acid (methionine) synthesis via METE or METH, nucleotide synthesis, and broader methylation patterns, was next investigated in C. reinhardtii and P. tricornutum. Observed were higher differential expression of C1 cycle transcripts in both C. reinhardtii and P. tricornutum metE mutants under vitamin deprivation, which may be a result of altered metabolic capabilities. Comparing METE genes between related species in C. reinhardtii and P. tricornutum revealed that Edaphochlamys debaryana and Fistulifera solaris have two METE genes. The B12 dependent C. reinhardtii was able to form a mutualism with B12-producing bacteria Mesorhizobium loti and Rhodospirillum rubrum. An RNA sequencing experiment was conducted with samples taken just after initiation of a co-culture and then after subculturing once. Clean reads ranging from 53-81 million were obtained with ~95% coverage in all samples. Differentially expressed genes (DEGs) were found, including in ‘biosynthesis of secondary metabolites’, ‘oxidation-reduction process’, and ‘transporter activity’. Of note was that distinct differential expression patterns were seen in co-cultures with M. loti versus R. rubrum, and a distinct B12 deprivation response was apparent in the latter. B12-dependent P. tricornutum in co-culture with B12 producing Halomonas sp. did not form a stable mutualism. Comparative analysis of the transcriptional regulation of METE in C. reinhardtii and P. tricornutum was carried out by using respective METE promoters (PMETE) to drive expression of fluorescent reporter mVenus. The findings showed crucial differences between the reporters in terms of B12 dose response, timing, and repression via B12 analogues. When the reporter strains were co-cultured with B12-producing bacteria M. loti repressed expression of mVenus in C. reinhardtii but Halomonas sp. did not in P. tricornutum, indicating that B12 was not supplied to the alga in the latter case. Diurnal datasets were also investigated and found that METE in C. reinhardtii is under strict diurnal control, whereas the P. tricornutum METE exhibits higher expression through the dark cycle. However, when P. tricornutum PMETE-mVenus reporters were assayed over the diurnal cycle, there was no change in fluorescent expression, suggesting the diurnal regulation may not be through the promoter.
Open Access
Optimising fungicide application tactics: using mathematical modelling to understand qualitative and quantitative fungicide resistance management
Taylor, Nick
Plant diseases pose a significant threat to food security, and fungal plant pathogens can be particularly damaging due to enormous evolutionary potential. Two major control strategies for fungal pathogens include chemical control with fungicides and use of disease-resistant cultivars. Both strategies are threatened by pathogen evolution leading to fungicide resistance and loss of cultivar efficacy (‘cultivar breakdown’). Both fungicide resistance and cultivar breakdown can be characterised as qualitative or quantitative, depending on the genetic basis for resistance/breakdown. Mathematical models have an important role in understanding fungicide resistance and cultivar breakdown, and modelling studies offer numerous advantageous over experimental studies. We present a model of qualitative fungicide resistance and one of quantitative fungicide resistance and cultivar breakdown. The model of qualitative resistance extends a model from the literature, while the quantitative resistance model is entirely novel. Both models are parameterised to address Septoria, the most prevalent disease of wheat. Although qualitative resistance has been widely studied, many single-site fungicides (i.e. those challenged by qualitative resistance) face widespread resistance, meaning that quantitative resistance is increasingly important. However, the mechanisms underlying quantitative resistance/breakdown are more complex and suitable data for model fitting is harder to source. We present the first model of quantitative resistance to be fitted to field data for both fungicide resistance and cultivar breakdown. Fungicide mixtures can help delay resistance. Although many studies focus on mixtures of fungicides to which resistance is qualitative, the optimal strategy is not characterised if the initial resistance frequencies (RFs) to two fungicides in a mixture differ. Past work showed that equal selection for single resistant strains in the first year was the optimal strategy when initial RFs were equal but did not consider when the initial RFs differ. We show that this strategy is often sub-optimal if the initial RFs differ and present an alternative strategy based on equalising RFs in the breakdown year. We test the robustness of this strategy to changes in parameters controlling fungicide efficacy and pathogen epidemiology, including between-season pathogen sexual reproduction. Previous modelling studies of Septoria neglected pathogen sexual reproduction, for simplicity, despite evidence that sexual reproduction is an important part of Septoria’s life cycle. We use the quantitative resistance model to determine how the number of fungicide applications per year affects resistance development, disease severity and yield. We consider how the optimal strategy varies depending on the time-frame of interest, before exploring how cultivar and fungicide control can be optimally combined to control the pathogen whilst minimising degradation of host and fungicide. We explore the effect of fungicide dose and compare the recommendations from the quantitative resistance model to those from the qualitative resistance model. Most fungicide resistance studies consider strategies which are fixed in time – i.e. rely on the same tactic used every year until failure. We present a flexible approach to optimise time-variable strategies, based on dynamic programming. This allows us to address an otherwise computationally infeasible problem to find the optimal strategy when time-variable strategies are permitted. We compare the improvement offered by the optimal time-variable strategy to the optimal fixed-time strategy.
Embargo
The Spatiotemporal Dynamics of Nutrient Exchange at the Arbuscule
McGaley, Jennifer; McGaley, Jen [0000-0002-5191-2005]
Plants are the major primary producers, source of biomass, and defining feature of ecosystems on Earth. But their ability to fix carbon is only half the story: plant growth also requires a suite of mineral elements that usually exist at limiting-levels in the soil. Most land plant species rely on a symbiotic partnership with arbuscular mycorrhizal (AM) fungi to meet this nutrient demand. AM symbiosis involves the fungus-to-plant transfer of phosphorus and nitrogen, and the reciprocal plant-to-fungus transfer of carbon at specialised structures within plant root cells, the arbuscules. While many studies have evidenced the AM-mediated transfer of these nutrients, the spatial and temporal elements remain largely unknown. This is significant because AM symbiosis is highly dynamic and heterogenous, from the scale of a single arbuscule up to the level of a colonised root system. This PhD work therefore aimed to uncover the spatiotemporal dynamics of nutrient exchange at the arbuscule during AM symbiosis. Fluorescent reporter lines in rice and novel microscopy techniques were developed to follow the localisations of AM-specific nutrient transporter proteins throughout arbuscule lifetime. Reporters for OsPT11, responsible for fungus-to-plant phosphate transport, revealed highly specific expression-timing and localisation, largely coinciding with arbuscule branching, which are essential for functional AM symbiosis. Surprisingly, OsAMT3;1, responsible for ammonium transport in the same direction, showed a longer window of expression and broader spatial distribution, suggesting the absence of a universal time or domain of nutrient exchange. This was augmented by a further set of unique dynamics shown by the lipid transporters OsSTR1 and OsSTR2, responsible for the opposite direction of transport. OsSTR1/OsSTR2 localised to the arbuscule earlier than OsPT11, but lipid export was not essential for OsPT11 or OsAMT3;1 expression. Variation in relative abundance of each nutrient transporter at the arbuscule was consistently observed, and was found to be responsive to plant nutrient status. Together, these results paint a picture of distinct co-ordination of symbiotic phosphorus, nitrogen, and carbon transporters, with arbuscules representing functionally unique structures as opposed to identical units of nutrient exchange. Additional to answering biological questions, the micrographs produced during this work were re-purposed to engage the public with AM symbiosis. The final chapter of this thesis discusses the potential of scientific visual data to increase awareness, appreciation, and knowledge of ‘overlooked organisms’, combining examples from the literature with outreach work carried out during this PhD. The resulting benefits are highlighted, providing inspiration and motivation for researchers to share their images beyond academia.
Open Access
Evolutionary perspectives on plant responses and sensitivity to environmental change
Thomas, Anne; Thomas, Anne [0000-0002-2808-6462]
Global change is putting unprecedented pressure on plants to adapt or migrate to avoid extinction. Studying the past responses of plants to environmental change can shed light on the potential evolutionary outcomes and sensitivity of species to future environmental change. These processes are especially relevant to highly diverse, evolutionarily rich, and ecologically vulnerable alpine ecosystems. My PhD aims to narrow the uncertainty about how plant lineages with a range of lowland and alpine species will be impacted by global change by studying the historical biogeography, trait and species diversification, and ecological strategies of alpine species in a phylogenetic framework. Chapter 1 reviews current knowledge about the relative roles of migration and adaptation in plant responses to climate change and how historical biogeographical and evolutionary modeling provide novel insights to these questions. Chapter 2 applies recent developments in sequencing methods to construct a new, near-complete phylogeny of a diverse species radiation, New Zealand Veronica, also addressing questions about how to resolve difficulties in reconstructing phylogenetic relationships in recent, rapid radiations such as Veronica. This group serves as an important case study for further evolutionary questions about the relationships between habitat, species diversity, and environmental change. Chapter 3 estimates the contributions of in situ cladogenesis (i.e., the formation of new species) and colonization from lowland habitat in generating mountain diversity in Veronica. Further, the chapter explores the importance of niche adaptation and divergence in contributing to cladogenesis, and presents a general, conceptual model to understand how mountain diversity accumulates. Chapter 4 compares the potential range and niche change required for plant species to respond to future climate change relative to the change undergone since the mid-Holocene. It also determines which niche traits can predict “winners” and “losers” under climate change. Chapter 5 discusses the main findings of the thesis and ends with proposed avenues for future research.
Open Access
How growers make decisions impacts plant disease control
Murray-Watson, Rachel; Murray-Watson, Rachel [0000-0001-9079-5975]
Whilst the spread of plant disease depends strongly on biological factors controlling transmission, epidemics clearly also have a human dimension. Disease control depends on decisions made by individual growers, who are in turn influenced by a broad range of factors. Despite this, human behaviour has rarely been included in plant epidemic models. This thesis focusses on addressing this oversight by developing combined epidemic and economic models of disease spread. We use simple continuous-time models of disease spread which we couple with behavioural models which set the management programme of the growers for the next growing season. Our models are rooted in game theory, with growers making strategic decisions based on the expected profitability of different control strategies. In the first instance, we compare different versions of this behavioural model, which differ in terms of the information used by the growers to assess profitability. We investigate these models in the context of Cassava Brown Streak Disease (CBSD) and its management via the use of clean seed systems (CSS). We find that both the information used by growers to assess profitability and the perception of economic and epidemiological parameters influence long-term participation in the CSS. Over-estimation of infection risk leads to lower participation in the CSS, as growers perceive that paying for the CSS will be futile. Additionally, though the CSS can reduce the burden of disease, and allow a scenario in which all growers control, disease is not eliminated from the system. For the remainder of this thesis, we use one behavioural model to investigate the deployment of crop that is either resistant or tolerant to Tomato Yellow Leaf Curl Virus (TYLCV). We find that when growers used resistant crop, higher yields were achieved by both controllers and non-controllers, though widespread use of resistant crop was not achieved. The use of tolerant crop reduced the yields for non-controllers, but generally benefited its users, inducing a positive feedback loop that resulted in a high proportion of growers using tolerant crop. By extending this TYLCV model to allow a three-way choice of tolerant, resistant, and unimproved crop, we see again how growers prefer tolerant crop. However, these responses can be manipulated by changing the cost of each crop type through subsidisation schemes. To do this, we consider the efforts of a ``social planner" who moderates the price of crops. We find that subsidising tolerant crop costs the social planner more in subsidies, as its use encourages selfishness and widespread adoption. Subsidising resistant crop, however, increased the use of resistant crop, again enabling higher yields across the community of growers. Many of our results obtained were robust to spatial and stochastic effects. Some differences arose when growers narrowed their information sources to only consider those growers whose fields are in close proximity to their own, as this allowed assessments of profitability to be based on local disease pressures. In this thesis, we show how simple models of grower behaviour can be incorporated into both deterministic and spatial-stochastic models of disease spread. Understanding the influence of economic and epidemiological factors, as well as the feedback loops induced by different control mechanisms, on these behaviours can help to promote better outcomes for growers.
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The Carbon Density and Species Diversity of Myanmar's Tropical Forests
Tun, Kyaw Sein Win
Tropical forests play a vital role in climate change because they may act as sinks or sources of CO2 depending on their management and responses to climate. Satellite imagery indicates that 3.8% of Myanmar's forests were lost between 2001 and 2020, but the extent to which commercial forestry is degrading the remaining forests is not well established. Although Myanmar’s forests provide a range of economic, environmental, social, and cultural benefits, detailed knowledge of the country’s forest resources is still lacking. My thesis focuses on evaluating how carbon storage and tree species composition in Myanmar's tropical forests are affected by its management history and environmental factors. Chapter 1 introduces the ecology of Myanmar’s forests. It describes historical and current forest management systems and the nation’s commitment to international climate change mitigation interventions. Chapter 2 evaluates the drivers of aboveground carbon density (ACD) variation across the nation’s forests using a national ACD map derived from data collected by NASA’s space-borne LiDAR sampler (GEDI). Using matching analyses, I find that terrain (elevation, slope, and aspect) and climatic attributes (including precipitation) are positively related to ACD, plateauing at higher values. ACD is negatively affected by anthropogenic factors, declining close to cities and roads. Strictly protected forests store twice as much carbon as less regulated forests, indicating that establishing protected areas in Myanmar is an effective strategy for protecting high-carbon forests. Chapter 3 compares remotely sensed biomass estimates of ACD with traditional forest inventory datasets, using a government-funded national forest inventory (NFI) covering 320,000 km2 of dry tropical forests. I use the same statistical approach as employed in Chapter 2 to predict the influences of biophysical factors on ACD. Biophysical factors influence field estimates of ACD in qualitatively the same way as found using LiDAR data in Chapter 2. However, field estimates of ACD are about 1/3 the value of estimates based on remote sensing data iii and estimates from inventory plots and GEDI are found to be only weakly correlated even when data are averaged within municipalities to reduce the effects of local noise. This difference has probably resulted from human error associated with forest inventory, such as misidentification of tree species, using different sampling methods, and omitting trees from the inventory, but more work is required to understand why there are strong discrepancies. Chapter 4 analyses tree species distribution patterns, as this information is required for effective forest management yet is lacking at the national scale. Annual mean temperature, temperature in the wettest season, temperature in the summer, and precipitation in the driest season are found to be important climatic determinants of species composition. I conclude that the deciduous forests of Myanmar, which have been extensively selectively harvested for valuable timber, have the potential to sequester large quantities of carbon and protect tree diversity if adequately protected. Chapter 5 explores the effectiveness of a protected area at maintaining different forest types and conserving biodiversity. Focusing on forest inventory data I collected at the Popa Mountain Park, I evaluate biophysical influences on ACD on tree species composition and carbon. Elevation positively correlates with ACD, but species richness does not. Non-Metric Multidimensional Scaling analysis reveals that elevation is the most important determinant of species diversity in the park. Additionally, comparisons of ACD within the park and matched production forests across the entire country show that the park stores ten times the amount of ACD compared to the exploited production forests.
Embargo
Evolution and Function of Receptor-like Kinases in Arbuscular Mycorrhizal Symbiosis
Chiu, Chai Hao
Evolution of the nutritional mutualism between plants and arbuscular mycorrhizal (AM) fungi was crucial to evolution of life, as it enabled the conquest of the green lineage and its ecosystem on land. Today, apart from AM fungi, plants co-exist with a plethora of micro-organisms, and exactly how the plant immune system balances the need for defence against detrimental micro-organisms and beneficial ones such as AM fungi remains enigmatic. This work characterised plant plasma membrane receptors that perceive microbial carbohydrate patterns, and how they bring about cellular and developmental changes. To understand the origins of the Lysin-motif (LysM) receptor-like kinases (RLKs) and Symbiosis Receptor Kinase (SYMRK), comprehensive phylogenomic analyses of these receptors in more than a hundred plant genomes revealed their deep origins in green algae. I then defined the major clades of LysM-RLKs, their correlation with plant symbiotic capabilities, how the pseudokinases have diversified and refined, and how the ancestral LysM-RLK gave rise to the repertoire in extant land plants. Independent events involving domain fusions of LysM with kinase domains in the plant kingdom and beyond demonstrate how modularity of protein domains enable genetic innovations in the tree of life. A clearly defined receptor phylogeny also enabled systematic genetic dissection of the RLKs. Functions of four different receptor clades were carefully analysed in rice using single and double mutants. Careful assessment of AM colonisation in these mutants reveal chitin and lipochito-oligosaccharides as redundant signals, perceived by an equally redundant set of RLKs, as well as a symbiosis-specific receptor that represents a second checkpoint of symbiotic scrutiny – confirming insights from phylogenomic analyses. Perception of AM fungi also result in plants producing more lateral roots, a response observed for decades. Here, molecular mechanisms of this developmental response are elucidated, where general perception of chitin-derived molecules through LysM receptors and SYMRK activate lateral organ production. This response is also conserved in non-AM hosts, revealing the existence of an ancestral signalling function of these RLKs in shaping plant development – which may have been recruited in the evolution of nitrogen fixing root-nodules. Transgenic lines containing tagged-RLKs were also characterised, paving the way for future biochemical experiments to understand how the receptor complexes activate downstream signalling components for their biological functions.
Embargo
Petals, pigments and pollinator preferences
Symington, Hamish; Symington, Hamish [0000-0002-5360-7979]
As the world population rises, we will need to grow more food. Insect pollination is essential or beneficial for ~75% of crops, including most fruit, nuts, and commodity crops such as coffee and cocoa, yet there is evidence of significant global decline in insect numbers: this conflict is central to my research. Solutions involve developing self-fertile cultivars (time-consuming and not always possible), large-scale increase of pollinator numbers (possible but involving significant input or change over large areas), or the production of new cultivars which are more attractive to insects and which provide them with more or better nectar and pollen rewards for visiting (the basis of my research). With this latter approach, it will be possible to not only use the existing insect resource more efficiently, but also to increase the available food supply for pollinators, helping to boost their populations. Focussing on strawberries, an important commercial crop whose quality and yield are increased by insect pollination, I have measured nectar and pollen rewards in 20 commercial varieties of strawberry, identifying variation in nectar production based on temperature in some varieties, and variation in pollen quantity and viability. Using an image-recognition program I wrote, I have characterised variation in floral shape for the same varieties, identifying significant variation between varieties in flower size and shape. In our bee behaviour laboratory, I have used plastic artificial ‘flowers’ and lab colonies of bumblebees to test bee responses to the limits of observed variation in shape, in terms of recognition and finding speed, and found that more stellated flower outlines result in a small but non-significant decrease in the time it takes bees to find the 'flowers’. I have conducted experiments to measure bee feeding and vomiting rates when provided with nectar of differing sugar concentrations, which informs models which predict the sucrose concentration within nectar which maximises energy return to the nest. I have also tested bee decision-making when faced with rewards with varying ease of access, and found that bees can trade off the sugar reward within nectar against the energetic cost of accessing that nectar. These experiments help to inform efforts to produce crop varieties which maximise the rate of energy return to the nest. Finally, I have shown that previous observations of the nectar preferences of bumblebees conducted at laboratory scale are relevant at nectar concentrations and quantities found naturally in crop plants. Another floral trait which is highly relevant to pollinators is floral colour. Parallel to the strawberry research, I have been investigating the molecular mechanism of floral colour production in both the model plant Arabidopsis thaliana (which has white flowers) and a pink-flowered species in the closely related genus Aethionema. I have produced transformed Arabidopsis lines which overexpress pairs of transcription factors, which give information on which combinations produce the most pigment in that species. Additionally, using a comparative transcriptomic approach, I have identified transcriptional activators which are likely to be implicated in flower colour in Aethionema. However, experiments to ectopically express these activators in Nicotiana tabacum have not shown any increase in petal colour, indicating that the promoter sequences from Aethionema are not sufficient to drive heterologous expression.
Open Access
Meristem regulation in the early divergent land plant Marchantia polymorpha
Rebmann, Marius
Meristems are key features of land plant development, enabling post-embryonic organogenesis through precise spatial patterning of cell division and differentiation. Extensive work on flowering plant meristems has revealed gene circuits driving meristem patterning, which are increasingly targeted to engineer crop development. However, genetic redundancy and morphological complexity present powerful obstacles to explore engineering of meristem regulation. Marchantia polymorpha is an early divergent land plant which has received re-surging interest as a model plant, following the publication of its genome which revealed extraordinarily low levels of genetic redundancy. Marchantia’s genetic simplicity is mirrored by a simple body plan. Asexual propagules called gemma are a particularly attractive model to study meristem regulation owing to their small size, disc shaped morphology and open development. This permits facile live imaging of early development of whole plants at cellular resolution. Despite these benefits, our understanding of the Marchantia meristem remains rudimentary compared to other plant models. This dissertation describes the use of single cell RNA-sequencing and novel marker lines to define the cell composition of the Marchantia meristem and the use of genetic and experimental perturbation to interrogate the gene networks and phytohormone patterning systems governing meristem maintenance and initiation. I describe the use of a proximal promoter library comprising a near complete collection of promoter elements for all Marchantia transcription factors, to identify novel meristem markers. Fluorescent reporters for approximately 20% of all Marchantia transcription factors were screened in gemma, identifying novel cell type markers. I present the analysis of a single cell RNA-sequencing dataset comprising approximately 7’000 cells from developing gemmalings. I characterise Marchantia cell types based on their transcriptomic profiles and identify corresponding cell identities in vivo using marker genes. The data captured broad developmental gradients of proximal-distal and dorsal-ventral patterning as well as resolving specific cell lineages such as rhizoid cells at unprecedented resolution. I define central stem cells as tissue organisers of the Marchantia meristem. I show that central stem cells are auxin sources and develop novel markers to study auxin response in gemma. I identify a ERF/AP2 transcription factor Mp ERF20 as a positive regulator of central stem cell fate. I interrogate the regulation of the division zone by cytokinins and identify Mp CYCD1 as a key regulator of cell division rates in the Marchantia meristem. I show that Mp CYCD1 overexpression can be leveraged as a tool to induce ectopic cell divisions. I characterise meristem regeneration in Marchantia explants using marker lines and precise surgical manipulation. I observe the activation of proliferation markers and establish auxin transport reorganisation as a critical driver of meristem regeneration. Using this data I propose a model for meristem maintenance and regeneration in Marchantia which will form an important framework for future attempts to engineer growth in this simple morphogenic system.
Embargo
Strategies to increase production of betalain pigments and their use as SynBio tools
Timoneda Monfort, Alfonso
Betalains are plant pigments showing a wide range of potential applications in the pharmacological, biotechnological and commodity sectors. They are currently being used as natural food colorants, and intermediates of the biosynthetic pathway exhibit important pharmacological properties (e.g. ʟ-DOPA). Due to this, there is an increasing interest in improving existing plant sources and obtaining new bio-industrial methods for semi-synthetic production. Betalain biosynthesis is achieved in plants via a relatively short metabolic pathway which has been successfully engineered in a diversity of heterologous hosts including plants, bacteria and yeast. Here, we explore a variety of mechanisms to utilise betalain pigments and enhance their bioindustrial production. First, we generated a betalain-based reporter system to visualise arbuscular mycorrhiza symbiosis processes in in $\textit{Medicago truncatula}$ and $\textit{Nicotiana benthamiana}$ roots. By expressing the betalain biosynthetic genes under plant symbiosis specific promoters, we were able to effectively restrict betalain production to areas of the root engaging with fungal colonisation. We also showed that this is an efficient tool for the dynamic tracing of root colonisation $\textit{in vivo}$ over time. This presents an advancement from the current tools used for the visualisation of fungal structures by the symbiosis research community and demonstrates the potential of the betalain pathway for the efficient reporting of other plant physiological processes. Secondly, we explored the existing natural variation in betalain biosynthetic enzymes in order to identify variants exhibiting higher activity than those currently known that could be used to improve betalain production. Initially we focused on the arogenate dehydrogenase (ADH) enzymes in the Caryophyllales, which show relaxed sensitivity to feedback inhibition by product and can produce higher titres of tyrosine, the substrate of the betalain pathway. We interrogated the ADH phylogeny of flowering plants to identify putative ADH homologs with relaxed sensitivity, and found that this phenomenon is not restricted to ADH enzymes of the Caryophyllales, and has likely also occurred repeatedly in other phylogenetically distant clades. Thirdly, we explored the utility of enzymatic diversity held within different origins of betalain pigmentation. Purification and analysis of different ʟ-DOPA 4,5-dioxygenase (DODA) enzymes from different inferred origins in the Caryophyllales revealed distinct kinetic properties and differences in overall pigment production performances in $\textit{E. coli}$ cultures. Scale-up experiments with the DODAα1 enzyme from $\textit{Carnegiea gigantea}$ (Cactaceae) in 2 L and 30 L bioreactors allowed for the biotechnological production of unsurpassed titres of betalains. Finally we used ancestral sequence reconstruction and enzyme activity assays in yeast to identify the residues important for high DODA activity in the Globular Inclusion clade, containing the Cactaceae. With this approach, we also confirm that the Globular Inclusion clade represents an independent origin of betalain pigmentation in the Caryophyllales, distinct from other known betalain-producing taxa such as $\textit{Beta vulgaris}$.
Open Access
The hormonal regulation of PIN protein localisation and its relevance to shoot architecture
Bridgen, Anthony John
Shoot branching plasticity relies on integration of diverse signals to regulate the activity of buds, which is partially mediated through auxin transport by PINs and the regulation thereof. Whilst PIN behaviour has been well characterised, the mechanisms by which PINs sense and respond to auxin, strigolactone and cytokinin remain unknown. In this thesis I investigate the regulation of PIN polarity in response to these hormones, presenting evidence of an age-dependent aspect to PIN1 behaviour. I then demonstrate that NPA-sensitive auxin flux or auxin concentration are insufficient to explain PIN1 behaviour and attempt to identify a mechanism by which PIN1 senses and responds to auxin at a sub-cellular level. Following this, I demonstrate the requirement of the central region of the PIN1 hydrophilic loop to confer strigolactone sensitivity and characterise the effect of the loss of this response on plant phenotypes and bud growth dynamics. Finally, I demonstrate cross-species differences in PIN hormone responses. As a whole, this work advances our understanding of hormonal control of auxin transport in the shoot and the way in which this affects shoot architecture.
Open Access
Engineering a Reverse C4 Photosynthetic Pathway in C3 species Arabidopsis thaliana
Di, Zhengao; Di, Zhengao [0000-0002-5085-5774]
C4 photosynthesis is a specialised carbon concentrating mechanism that achieves higher rates of photosynthesis. Engineering C4 photosynthesis into C3 plants could improve agronomic features of staple C3 crops. Introducing the biochemistry of C4 photosynthesis is considered a key step in engineering the C3-to-C4 transition. Herein, a reverse C4 photosynthesis system that aimed to install C4 biochemistry in the opposite cell types from those used by C4 species was investigated. This approach was hypothesised to replicate the spatial patterning of the C4 biochemical pathway but make use of the native leaf anatomy of C3 species. The main objective of this thesis was to test if introducing a reverse C4 photosynthetic pathway in C3 Arabidopsis thaliana could improve its photosynthetic efficiency. Two subtypes of the C4 biochemical pathway, the PEPCK and NAD-ME subtypes were tested. A. thaliana genes most suitable for assembling these subtypes were selected based on homology to C4 genes and relative transcript abundance. Transgenic plants carrying a reverse PEPCK C4 pathway displayed no differences in carbon isotope composition or Photosystem II activity, and in a genetic background with less RuBisCO showed lower CO2 assimilation. Genes for a reverse NAD-ME C4 pathway were assembled into two large multigene constructs containing 9 and 10 transcription units. Homozygous lines carrying each construct were obtained and showed overexpression of most transgenes. Although plants carrying bundle sheath genes of the pathway showed no visible phenotypes and those carrying mesophyll genes showed retarded growth, the latter phenotype seemed to be alleviated in F1 crosses containing genes encoding a full reverse NAD-ME C4 pathway. Work is also reported that aimed to explore two alternative approaches to accelerate the main objective of C4 engineering. First, clustering enzymes of a metabolic pathway via protein scaffold was tested. Three enzymes for the biosynthesis of betalain were transiently expressed in tobacco in the presence of scaffold proteins of various structures. However, betalain production was inhibited. Varying the number of domains on scaffold proteins or attaching them to the vacuole membrane did not generate significant impact on pathway efficiency. Second, a microdroplet-based cell-free protein expression system was built to mimic plant metabolic pathways in vitro. Fluorescent proteins and enzymes could be directly synthesised from DNA templates in microdroplets. Fluorescence corresponding to betalains was observed when part of the pathway was expressed and substrate provided. A potential future application of this system was to mimic multicellular physiological processes such as the C4 biochemical pathway.
Embargo
Understanding the development, evolution, and function of bullseye pigmentation patterns in Hibiscus trionum
Fairnie, Alice
Colourful spot, stripe and ring patterns decorate the corolla of many flowering plants and fulfil important biotic and abiotic functions. These petal patterns are created by spatial differences in pigmentation, cell shape and texture of the adaxial petal epidermis. The mechanisms controlling formation and evolution of these patterns, and their exact role in plant-animal communication are not well understood. My PhD adds to current understanding of petal patterns by investigating the development, evolution, and function of the bullseye pigmentation pattern on Hibiscus trionum petals. The bi-coloured bullseye pattern of H.trionum is created from contrast in cell shape, cuticle texture, and pigmentation between the bottom and top of the petal. The bottom petal appears shiny and purple because cells in this region are elongated with a striated cuticle, and are pigmented with anthocyanins. A minimal regulatory network restricting anthocyanin pigment production to the bottom of Hibiscus trionum petal was identified. The network relies on two MYB regulators: HtCREAM1 which represses anthocyanin production in the top of the petal; HtBERRY1 which promotes anthocyanin biosynthesis in the bottom of the petal. This minimal network is a starting point to understand the molecular mechanisms both creating, and creating diversity, in petal patterns. Natural variation in the bullseye pattern in close relatives of H.trionum from Australia and New Zealand is in part due to four independent restrictions of anthocyanin pigmentation in the petal bottom. Preliminary results suggest restriction of pigmentation in H.richardsonii, sister-species to H.trionum, results from mutation in the regulatory region and the coding sequence of the BERRY1 homolog of H. richardsonii. Natural variation in the bullseye pigmentation pattern could reflect a function in plant-pollinator communication. Buff-tailed bumblebees were found to discriminate between, and prefer, artificial flowers with H.trionum-like bullseye patterns to H.richardsonii-like bullseye patterns.
Embargo
Fostering Populations of Arbuscular Mycorrhizal Fungi Through Cover Crop Choices and Soil Management
Crane, George
Over 70% of land plants, including many key agricultural crops, form a beneficial, symbiotic relationship with arbuscular mycorrhizal (AM) fungi. This has triggered interest in the potential role of these fungi in sustain-able food production for an increasing population. However, it is known that many common farming practices can negatively influence both the diversity, and abundance of the AM fungi. It is therefore desirable to identify farming practices or amendments that can foster these fungal populations to increase crop and soil benefits, including yield. Cover cropping, the growing of non-food crops outside of regular crop produc-tion for the role of protecting and improving soil, has also been suggest-ed to influence both the diversity and abundance of AM fungi. A large-scale analysis of AM fungal diversity in UK agriculture provided a framework for further analysis of how cover crops, and soil amendments influence AM fungal communities. Replicated trials in both glasshouse and field conditions have shown evidence that multiple iterations of cover crops can increase the extent to which plants are colonised by AM fungi, although this had no measurable impact on yield. In the same trial, it was shown that long term application of nitrogen fertiliser influenced AM fungal community composition, but this observation was not made in a shorter validation experiment conducted at the field scale. In a sepa-rate trial, addition of a commercial AM fungal inoculum had little impact on the AM fungal community, crop growth, or yield in field conditions, further suggesting that multiple iterations of soil amendments are re-quired to cause measurable, long-term shifts in AM fungal diversity and benefit.
Open Access
Modelling the impact of Phytophthora austrocedri on UK populations of native juniper (Juniperus communis s. l.)
Donald, Flora; Donald, Flora [0000-0001-6266-9189]
Introductions of non-native plant pests and pathogens are increasing, negatively impacting natural environments. Mitigation requires knowledge of the drivers of pathogen introduction, establishment and spread, rarely available when pathogens first emerge in novel settings. This thesis uses multi-scale ecological modelling to understand environmental and land management factors driving patterns in infection and impact of the newly discovered oomycete pathogen, Phytophthora austrocedri, on juniper (Juniperus communis). I first surveyed potential abiotic and biotic drivers of disease severity across three, geographically separate juniper populations with different infection histories. In all populations, disease severity increased with increasing soil moisture. Associated plant species that could be used to locate microsites at higher risk of infection were also identified. Change in infection intensity during a four-year period was then mapped across a single juniper population and related to environmental factors underpinning the presence and density of juniper and driving P. austrocedri spread. Colonisations usually occurred within a ~500m radius of previously symptomatic trees, with infrequent dispersal beyond 1km, potentially mediated by livestock and deer. By compiling a novel dataset, I revealed larger, more frequent supplementary juniper planting events increased the likelihood of P. austrocedri presence. Stakeholders managing, monitoring, and growing juniper then participated in a survey investigating how practitioners consider disease risks and whether these processes could be better supported by decision tools. Lastly, a machine learning model and risk map was developed that predicted juniper populations in northern England and central Scotland are at highest risk of infection due to acidic soil pH and increased roe deer density. My research demonstrates how incorporating a wider range of abiotic and biotic drivers, exploring scale dependence, and integrating stakeholder knowledge can improve the predictive accuracy of host-pathogen-environment models. The results are used to recommend strategies (e.g. reductions in grazing pressure, natural juniper regeneration and heightened on-site biosecurity) to mitigate the serious threat posed by the pathogen to UK biodiversity and habitat restoration goals.
Open Access
Investigating the role of the circadian clock in the physiology of wheat
Taylor, Laura
The circadian clock is an endogenous timing mechanism which acts to synchronise internal processes with daily and seasonal cycles in light and temperature. In Arabidopsis, the components which comprise the circadian oscillator and output pathways are well defined. Photoperiod dependent flowering, metabolism, growth and defence signalling networks receive information regarding the timing of the day/night cycle from the circadian clock. Many circadian output pathways are targets for crop improvement, with variation at circadian loci selected for during domestication and breeding. Investigations into how the circadian clock controls agronomically important traits may identify novel targets for breeders looking to create new elite wheat cultivars. The first aim of this thesis was to identify naturally occurring variation in circadian clock genes across wheat cultivars important in modern breeding efforts. In Arabidopsis, LUX ARRYTHMO (LUX) and EARLY FLOWERING 3 (ELF3) are circadian clock genes which are co- expressed at dusk, forming a transcriptional repressor complex which restricts the expression of day phased circadian clock genes. Deletions, which may result in a null allele, were identified in LUX along with presence absence variation (PAV) for ELF3 across different wheat cultivars. Sequence analysis of ELF3 and LUX promoter regions identified enrichment of motifs associated with morning and evening phased expression respectively, supporting previous studies which indicate that the phasing of LUX and ELF3 expression is not conserved between Arabidopsis and wheat. I present further tools to investigate the diel turnover of LUX and ELF3 proteins. The second aim of this thesis was to study the effect of perturbation to the circadian clock on circadian output pathways. Predicted loss of function of the circadian clock gene GIGANTEA (TtGIab) results in circadian arrythmia in chlorophyll fluorescence parameters and late flowering in long and short day controlled environment conditions. No differences in flowering time were observed in the field. Perturbation to the circadian clock through loss of functional ELF3 (TtELF3ab) and TtGIab resulted in mismanagement of diel carbohydrate turnover. The implications for growth and biomass accumulation are discussed. This thesis demonstrates that the wheat flowering and carbohydrate turnover pathways are controlled by the circadian clock. Utilising variation at circadian loci may provide novel targets for breeders looking to manipulate these systems for improved crop performance.