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Theses - Plant Sciences


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  • ItemOpen Access
    The interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins
    Crawshaw, Samuel
    The cucumber mosaic virus (CMV) 2b multifunctional protein is a suppressor of plant defences and an important determinant of viral pathogenesis. The effect of the 2b protein on symptom induction is due in large part to its physical interaction with the host RNA silencing factor Argonaute 1 (AGO1). The consequent inhibition of AGO1 activity induces developmental symptoms such as stunting and organ deformation in CMV-infected plants. In *Arabidopsis thaliana*, if 2b-induced inhibition of AGO1 is too strong this de-represses resistance against aphids, the insect vectors of CMV, which will be deleterious to virus transmission. In my initial work, I helped to establish that another CMV protein, the 1a replication protein, also binds to the 2b protein and modulates the 2b-mediated inhibition of AGO1 activity. This prevents induction of aphid resistance in CMV-infected *A. thaliana* plants and represents a novel regulatory system in which viral counter-defence protein functions are selectively modulated by another viral protein. I investigated why 2b proteins of Subgroup IA and IB CMV strains seemed able to interact with AGO1 and induce strong developmental symptoms and aphid resistance whereas 2b proteins of CMV strains of Subgroup II could not. Challenging established ideas, I demonstrated that a Subgroup II strain (LS-CMV) 2b protein can physically interact with and inhibit AGO1. I also found that the 2b protein of an atypical CMV Subgroup IA strain (Ho-CMV) that does not induce symptoms also binds to and inhibits AGO1. I showed that the property that distinguishes *in vivo* complex formation between AGO1 and the Ho-CMV or LS-CMV 2b protein is that it occurs predominantly in host cell nuclei. However, most Subgroup IA or IB CMV 2b proteins complex with AGO1 to a greater extent in the cytoplasm. I identified specific 2b residues that appear to facilitate 2b-1a protein-protein interactions, and in the process of investigating potential regions of the 2b protein needed for 2b-AGO1 complex formation I found evidence that the 2b protein may contain intrinsically disordered regions. Such disorder may explain the ability of the 2b protein to interact with a wide range of factors.
  • ItemOpen Access
    Using remote sensing to track resilience of subtropical rainforests against fires and tropical cyclones
    Chan, Aland HY; Chan, Aland H. Y. [0000-0001-6740-0680]
    The large-scale restoration of tropical and subtropical rainforests is crucial for mitigating climate change and biodiversity loss. Disturbances such as fire and wind potentially undermine efforts to restore degraded landscapes, interacting with the existing vegetation and background topography to produce complex patterns of damage. It is therefore crucial for us to understand these interactions and study the factors that contribute to disturbance resilience. Rapid developments in the field of remote sensing have provided new tools to study forest-disturbance dynamics across unprecedented spatiotemporal scales. In this thesis, a range of high-resolution remote sensing products, including aerial imagery, satellite multispectral imagery, and airborne LiDAR scans, were used to evaluate how fires and tropical cyclones have affected vegetation in wet subtropical Hong Kong. Chapter 1 provides an overview of how forest disturbances interact with restoration ecology. It then describes the vegetation history of Hong Kong, highlighting how the region represents an interesting case study as a long-running restoration project over highly degraded landscapes in the wet tropics. Chapter 2 reconstructs the fire history of Hong Kong using a 34-year Landsat imagery time series. Burn area detection in the wet tropics and subtropics is challenging due to high cloud cover and rapid revegetation of burn areas. A pipeline was developed to process hundreds of satellite multispectral images and accurately map out thousands of burnt areas. The pipeline additionally dated every detected burn area polygon and estimated burn severity for pixels in the burn area. The final product is the first of its kind in wet tropical Asia. Chapter 3 proceeds to use this burn area and severity time series to study fire-vegetation feedbacks in Hong Kong. When early successional vegetation is more fire susceptible than late-successional closed-canopy forests, positive fire-vegetation feedbacks are created. These feedbacks can then form “fire traps” that undermine restoration of degraded landscapes. Here, fire occurrence and post-fire recovery in different vegetation types were investigated. The results provided compelling evidence for the presence of strong fire traps in Hong Kong. Chapter 4 further expands on these results by investigating how landscapes can escape these fire traps. The results demonstrate that fire suppression increased forest cover in Hong Kong, and these changes could be accurately modelled by parameters estimated from remote sensing. To meet restoration objectives, land managers would benefit from explicit, quantifiable targets based on model predictions to ensure the restoration success is not undermined by changes in fire regimes under climate change. Chapter 5 describes a pipeline to model long-term mean and typhoon maximum wind speeds across the rugged topography of Hong Kong, as a precursor for Chapter 6. Specifically, wind models based on computation fluid dynamics (CFD) modelling were validated by wind data collected from a dense network of weather stations and our own anemometers. Chapter 6 analyses the resulting wind maps and a repeated LiDAR dataset (2010, 2017, 2020) to study forest resilience against strong tropical cyclones. The LiDAR dataset captured the forest damage incurred during Typhoon Mangkhut in 2018, which was the strongest tropical cyclone to affect Hong Kong in over 40 years. Plantations, tall forests, and normally wind-sheltered forests were found to be more susceptible to tropical cyclones. Effects of tropical cyclones and wind exposure cascaded through time to create strong wind-related limits on local forest height. Overall, this thesis provides a detailed account of the patterns of resilience against fire and tropical cyclones in the wet tropics. Such knowledge on resilience could help land managers better plan and restore degraded landscapes in the wet tropics under changing climate and disturbance regimes.
  • ItemOpen Access
    Stomatal regulation in C4 photosynthesis
    Bernardo, Emmanuel
    The productivity of crops is largely determined by how efficiently they convert carbon dioxide (CO2) into organic molecules through the process of photosynthesis. The exchange of gases between the atmosphere and the leaf intercellular spaces is facilitated by stomata, tiny pores found on the leaf epidermis. These pores act as gateways, allowing CO2 to enter the leaf while water vapor exits through transpiration. The regulation of stomatal behavior results in a trade-off between CO2 uptake and water loss in higher plants. While genetic and biochemical analysis of stomatal regulation has focused heavily on the C3 species *Arabidopsis thaliana*, it is well-known that stomatal regulation can vary between different species and different photosynthetic types. Therefore, this PhD project aimed to investigate stomatal regulation in C3 and C4 plants, comparing their responses to changes in light and CO2. To control for potential confounding effects not related to photosynthetic type, three phylogenetically-controlled species from Cleomaceae, *Flaveria*, and *Alloteropsis* were used in the study. Chapter focused on the response to blue and red light in C3 and C4 species. The study found that stomatal opening was highly responsive to blue light in a red light background in C3 dicot species of Cleomaceae and *Flaveria*, which confirmed previous observations in *Arabidopsis*. However, stomatal conductance in C4 dicot species was not responsive to the same treatment. In addition, stomatal opening in C3 and C4 species responded significantly to red light, while the intercellular CO2 was kept constant. However, this response appeared to be largely species-specific, rather than associated with photosynthetic pathway. The next chapter investigated stomatal regulation by [CO2]. To distinguish responses linked to photosynthesis vs responses driven by mitochondrial respiration, CO2 responses were assessed in darkness and under illumination with red light similar to growing conditions. Whereas the C3 species showed significant opening responses to sub-ambient CO2 in darkness, C4s were invariable. In contrast, the opening response to sub-ambient CO2 under illumination was stronger in the C4 species. Together with the differences in blue light response in Chapter 2, this may suggest that stomatal opening in the C4 species relies more on guard cell photosynthesis and less on mitochondrial respiration, compared to the C3 species. Finally, in the last chapter of the thesis, using NADP-ME antisense lines of C4 *F. bidentis*. It was hypothesized that perturbing the C4 cycle, resulting in reduced carbon flux into the bundle sheath cells, may alter stomatal regulation. The study found that mutant plants had significantly increased operating Ci compared to control plants. When Ci was kept the same as the control plants a significant increase in stomatal opening was observed in the mutant lines, suggesting that disruption of the C4 cycle disrupted stomatal sensitivity to CO2. Further exploration of molecular factors known to be involved in the guard cell CO2 response was performed, but turned out inconclusive. Altogether, the work in this thesis shows that while stomatal regulation in response to CO2 and light has remained qualitatively similar to the ancestral C3 pathway, quantitative shifts in sensitivity may have supported further optimization of CO2 assimilation and watervuse efficiency following evolution of the C4 pathway.
  • ItemOpen Access
    Understanding tropical forest dynamics through remote sensing and deep learning
    Ball, James; Ball, James [0000-0002-0165-5290]
    Protection of tropical forests is key to achieving global climate and biodiversity conservation goals. They play an essential role in carbon sequestration, water cycling, and nutrient exchanges, thereby regulating atmospheric composition and global climate patterns. However, they are under interlinked threats from deforestation and climate change, exacerbating biodiversity loss and potentially pushing these systems toward ecological tipping points. Computer vision techniques based on deep learning have emerged as potent tools for monitoring, conservation, and prediction efforts within these expansive and intricate ecosystems. This thesis uses these emerging technologies to understand forest dynamics at scales ranging from the phenology of individual tree crowns to large-scale deforestation. Chapter 1 introduces tropical forests, explores their ecological and societal value, and discusses the technological challenges and opportunities of studying them, with a focus on deep learning as applied to remote sensing data. Chapter 2 develops a tool to predict deforestation patterns based on convolutional neural networks (CNNs), working with freely accessible data to successfully forecast spatiotemporal patterns in the Southern Peruvian Amazon. Predicting the location of deforestation is difficult as it results from complex interactions within human-ecological systems but doing so may enable effective, adaptable prevention measures and conservation planning. The models, through their ability to discern deforestation drivers such as new access routes from remote sensing data, highlight the potentially transformational role of deep learning in conservation. In Chapter 3, I develop a new approach named *detectree2*, building on the Mask R-CNN architecture, which is capable of accurately detecting and delineating individual tree crowns from airborne RGB imagery taken over dense tropical forests. The foundation for any remote-sensing study of individual tree dynamics is accurate tree delineation. Trialled in diverse geographies, including Malaysian Borneo and French Guiana, I show this tool holds promise for large-scale forest studies. The performance of the detection and delineation, especially for tall trees, enables tracking of tree growth and mortality for the study of carbon dynamics from cheap, widely accessible photographic data. Chapter 4 develops a pipeline for identifying and mapping tropical tree species, building on the *detectree2* approach. This pipeline combines aerial photographic images taken every three weeks using a UAV with hyperspectral survey. Training and testing on a carefully crafted ground truth dataset, the two-step approach applies *detectree2* to multitemporal UAV-RGB data in order to automatically segment trees and then applies Linear Discriminant Analysis (LDA) to hyperspectral data to assign species. This new approach identified over sixty tree species with high confidence, achieving accurate species level mapping over 70 % of the total crown area of the landscape. Key to the improved mapping was the temporal stacking of imagery to delineate tree crowns accurately and a large, rigorously validated dataset of labelled tree crowns to train on. In Chapter 5, I use the data and techniques developed in the previous two Chapters to address ecological questions related to the phenology of tropical forests. Seasonal variation in canopy greenness has been observed from space, but the extent to which all species in diverse forests follow a similar pattern of leaf pigment changes, leaf flushing and loss remains unknown. I begin to address that knowledge gap by tracking phenology through drone-mounted sensors, providing a dataset that tracked individual trees in French Guiana at 3-weekly intervals over 34 months. 3,000 tree crowns were mapped and tracked using UAV LiDAR, revealing significant spatiotemporal variability in Plant Area Density (PAD) and distinct species-specific phenological patterns. By juxtaposing PAD with spectral metrics, I start to decipher variation in ``leaf amount'' and ``leaf quality'', offering some insights into how individual tree changes might impact forest productivity. Concluding Chapter 6 discusses ways in which integration of deep learning technologies and remote sensing into ecology research is helping to broaden understanding and conservation capabilities for tropical forests, by providing precise, scalable solutions spanning deforestation prediction, tree level monitoring, species identification, and phenological studies.
  • ItemEmbargo
    The role of SCLA-type GRAS transcriptional regulators in plant development and stress responses
    Hoey, David; Hoey, David [0000-0001-7848-0786]
    Sensing and responding to the environment is an adaptation which is essential to life. In biology, environmental stimuli and cues are sensed by receptors, which in turn activate signal transduction and downstream responses. Downstream responses are often mediated by transcriptional regulators, which can coordinate responses to environmental stimuli. GRAS-domain transcriptional regulators are a family of plant proteins, which are highly conserved across land plants and have diverse functionality. Many of these roles involve responding to environmental stimuli, and the signalling processes involving most GRAS proteins are fairly well understood in flowering plants. This thesis investigates the GRAS family in liverworts. Mp*GRAS7*, a GRAS gene in the liverwort *Marchantia polymorpha* (common name: Marchantia) was found to be transcriptionally responsive to stress, and it was hypothesised that it had a role in controlling biological responses to the environment. My phylogenetic analyses found that this gene belongs to a previously uncharacterized sub-clade of GRAS transcriptional regulators (SCARECROW-LIKE A), which is conserved across land plants but lost in the model plant *Arabidopsis thaliana*. In Marchantia, I found the gene to be transcriptionally responsive to abiotic and biotic stimuli, and also enriched in vegetative and generative reproductive organs. I have produced CRISPR knockout mutants in *Marchantia polymorpha* and its AM-compatible sister species, *Marchantia paleacea*, in order to investigate the functions of SCLA in liverworts. Phenotyping revealed Mp*GRAS7* to be involved in the vegetative to generative reproductive transition of *Marchantia polymorpha*. I also found SCLA to be a positive regulator of oomycete pathogen colonisation in both species. This thesis explores upstream regulation of Mp*GRAS7* by far-red light and drought stresses, as well as potential downstream targets. This investigation is the first characterisation of the functionality of this sub-clade of genes in any plant species, and thus contributes significant new knowledge to the fields of plant environmental signalling and development.
  • ItemEmbargo
    Early land plant origins of the MYB-bHLH-WDR complex
    Kongsted, Thea
    A major aim in the field of evolutionary developmental biology (evo-devo) is to identify the mechanisms by which developmental novelties arise. The observation that incited the present study was the identification of a common theme across the differentiation of multiple specialised cell types in the seed plant epidermis. Tripartite transcriptional complexes composed of a MYB transcription factor, a bHLH transcription factor and a WD repeat scaffold (MYB-bHLH-WDR or MBW complexes) regulate patterns of flavonoid pigments, trichomes and root hairs in this cell layer. We reasoned that investigating the origins and diversification of the MBW complexes may give insights into the origins of these cell types. Phylogenetic analysis and interrogation of the conservation of implicated sequence motifs led us to hypothesise that the MBW complexes identified in seed plants have a single origin in an ancestral land plant. This was associated with parallel duplication events in the *bHLH* and *MYB* families and recruitment of a more deeply conserved *WDR*. To test our hypothesis, we undertook functional analysis of orthologues from the bryophyte *Marchantia polymorpha*. We found that protein-protein interactions were conserved. Further, we found that putative MBW complex members in *M. polymorpha* phenocopied each other with respect to flavonoid pigmentation and specialised oil body metabolism. In conclusion, we infer that the functionally disparate MBW complexes of seed plants derive from a single MBW complex ancestral to land plants. Overlap between regulated processes in bryophytes and seed plants implies that the regulation of flavonoid biosynthesis, by targeting of early flavonoid pathway genes such as *Chalcone Synthase*, could have been the ancestral function. We find evidence that duplication of the *MYB* member has repeatedly been associated with neofunctionalization of the complex in rosids, for trichome and root hair patterning, and in liverworts, for specialised metabolism in a lineage-specific organelle, the oil body.
  • ItemEmbargo
    Engineering a cyanobacteriochrome two-component system into a synthetic, light-controlled gene expression system for plants
    Hofmann, Roberto
    Our inability to perturb the expression of genes at cellular resolution in planta has hampered our investigation of spatial and temporal dynamics. Ideally, we would like an orthogonal, genetically encoded system, that allows us to reversibly perturb the expression of genes of interest with maximal spatiotemporal resolution in a minimally invasive way. Our lab has reengineered the CcaS/R light receptor two-component system from cyanobacterium Synechocystis sp. PCC 6803 into a plant-compatible synthetic light-controlled gene expression system called Highlighter. This system was initially deployed in transiently transformed *Nicotiana benthamiana* leaves. However, challenges remained: an incomplete mechanistic understanding of CcaS light sensing, light response spectra overlapping with plant light signalling pathways, low engineering throughput, and most importantly the inability to use the system in stably transformed plants. Highlighter exhibits a novel blue light response not observed in wild-type CcaS/R without an obvious biochemical basis. Here I describe the use of structural modelling, spectroscopy, genetics, and functional assays to investigate how CcaS responds to blue light. I demonstrate by targeted mutagenesis, domain deletions and domain swapping, that the blue-light response is due to a not previously characterised LOV-like domain. Next, I applied this insight to engineering new versions of the protein with improved light response kinetics. Further, I describe advances in engineering Highlighter to function in *Arabidopsis thaliana* and generate stable transgenics. I aim to target GA20OX1, a gibberellin (GA) synthesis gene with the aim of modifying GA patterns visualised by the FRET-based biosensor nlsGPS1 developed by our group. To speed up *in planta* testing of new Highlighter variants, I developed new visual assays and worked to identify optimal light conditions for widespread deployment. Finally, I attempted to deploy a light-controlled Cas9-based programmable transcription factor *in planta*, that has recently been developed for use in mammalian cells. I demonstrate the ability of the system to induce gene expression in planta in the first step to developing the system into a viable optogenetic system for use in plants.
  • ItemOpen Access
    Developing a genetic toolkit for plant-parasitic nematodes
    Kranse, Olaf
    Nematodes belong to one of the most diverse phyla on the planet. Most nematodes are free living and feed on bacteria, fungi, and protozoa. Plant-parasitic lifestyles are relatively rare in terms of numbers, however, through their parasitism these nematodes have a substantial impact on agriculture. They are estimated to account for over 10% of the annual life-sustaining crop losses, costing the industry roughly 100 billion U.S. dollars per year. There are two major groups of plant-parasitic nematodes: endoparasites and ectoparasites. The endo-parasites are the most economically important, and consequently the most widely studied. Endo parasitic nematodes, e.g., cyst nematodes, spend most or in some cases their entire life within the host, and feed exclusively on living host tissue. Cyst nematodes alter the expression of a multitude of host genes, to coordinate the formation of a syncytial feeding-site. Loss in the ability to anipulate host genes required for the formation of this syncytium has a negative impact on parasitism: resulting in reduced nematode size and/or a reduction in the infection frequency. Given the intuitive pathways to impact from this fundamental understanding, there is considerable interest in the field to identify these, so called, susceptibility genes and the mechanisms by which they are manipulated. This thesis describes and discusses efforts to expand the genetic toolkit for the plant- and nematode-side of the interaction to accelerate the study of the pathology as a whole. The thesis is principally focused on the model cyst nematode *Heterodera schachtii* due to its ability to parasitise the model plant *Arabidopsis thaliana*. Knowledge on the nematode-side of the host-parasite interaction remains limited, partially due to the lack of functional genetic tools. Prior to this work, there was no method available to interrogate nematode gene “gain-of-function”, and only one method (RNA-interference) available to interrogate nematode gene "loss-of-function". The first experimental chapter details a body of work aimed to address this constraint. It describes various attempts to deliver and express foreign genetic material in plant-parasitic nematodes using liposome-based transfection. Ultimately, the first gain-of-function experiments are demonstrated for any plant parasitic nematode. Exogenous mRNA encoding eGFP and Luciferase are delivered to, and translated, in Nematoda. On the plant-side of the interaction, functional genetic tools are already well established. The challenge is phenotyping of parasitism. Historically, infection is quantified by eye under the microscope. The main limitations of this approach are: 1) the relatively small number of technically tractable phenotypes (i.e., number of nematodes); and 2) the laborious nature of quantification. The second chapter describes efforts to lift both constraints using custom 3D printed hardware and software to essentially digitise the assay. This new approach facilitates the measurement of infection, provides new phenotypes for analysis, and ultimately sets the stage for large-scale forward genetic screens. Finally, the ability of this new screening method to facilitate the identification of new S genes was demonstrated. An experiment was conducted to measure the transcriptional response of *A. thaliana* shoot infection by *H. schachtii*. These data were cross-referenced to a published dataset of the transcriptional response of *A. thaliana* root infection to define a tissue independent response to nematode parasitism. To identify new putative S genes, a screen of mutants of differentially regulated genes was conducted using the new phenotyping capability. Taken together, this work expands the tools available for the study of cyst nematodes demonstrating: 1) expression of exogenous genes in Nematoda; 2) digitisation of nematode phenotyping; and 3) identification of putative S genes by combining tissue-specific infection datasets.
  • ItemEmbargo
    Tracing the role of D14L as a regulator of arbuscular mycorrhizal symbiosis
    Hull, Raphaella
    Arbuscular mycorrhizal (AM) symbiosis between plants and AM fungi is conserved across the land plants and is the default nutrient uptake strategy for plants in nature. Plant recognition and accommodation of AM fungi requires signalling through two evolutionarily ancient pathways: the common symbiosis signalling pathway (CSSP) and the DWARF14-LIKE (D14L) pathway. D14L has diverse roles in plant development, including inhibition of mesocotyl elongation during seedling development and symbiotic perception of AM fungi in the soil. During D14L signalling in rice, the negative repressor SMAX1 is degraded and CSSP components are transcriptionally upregulated, indicating that the symbiotic function of D14L is to modulate the CSSP. Meanwhile, the transcription factor NSP2, which is implicated in AM symbiosis and is a target of the phosphate starvation response, has been found to operate upstream of D14L in Medicago. In this study, the functional conservation of D14L and its symbiotic role relative to the CSSP and NSP2 were defined in rice. To investigate the evolutionary history of D14L, trans-species complementation of the rice *d14l* mutant by *D14L* homologues from a range of AM and nonmycorrhizal plants was performed. This analysis confirmed the highly conserved nature of the D14L protein and revealed that D14L does not specify the mycorrhizal status of angiosperms. To assess the role of NSP2 and closely related NSP1 in AM symbiosis in rice, CRISPR mutants were generated and investigated for defects in fungal colonisation and mesocotyl development. It was found that both NSP1 and NSP2 are essential for symbiosis and mesocotyl development. Furthermore, the mesocotyl phenotype of *nsp* mutants could be rescued by application of D14L signalling chemicals, providing evidence that both NSP1 and NSP2 are upstream of D14L in rice. To examine the downstream components of D14L signalling, complementation lines of rice *d14l* with the CSSP component CCaMK were generated. It was found that a gain-of-function CCaMK could complement the *d14l* mutant, revealing that D14L signalling regulates the CSSP for AM symbiosis. These results place the D14L signalling pathway at the centre of NSP and CSSP function. Transcriptome analysis of gain-of-function CCaMK lines demonstrated that CCaMK upregulates the expression of potential presymbiosis genes and genes relevant to conditioning a plant for symbiosis. Overall, a model is proposed in which D14L signalling functions as a master switch that can integrate the plant nutrient status into a decision to rapidly launch or block symbiosis.
  • ItemEmbargo
    Expanding the molecular tools for the microalgae Chlorella vulgaris and Phaeodactylum tricornutum
    Hickland, Patrick
    Many species of microalgae have attracted attention from industrial biotechnology with the goal of harnessing specific physiological traits in applications including fuel, food, feed and high value medicinal products. However, to realise their full potential certain species will require optimisation through engineering. This study focussed on molecular tool development to address bottlenecks hindering the engineering of two microalgal species: the green alga *Chlorella vulgaris* and the marine diatom *Phaeodactylum tricornutum*, both exhibit rapid growth rates and high density cultures making them prominent candidates for use in numerous applications. Work in this thesis was sponsored by the algal biotechnology company Algenuity, Stewartby, Bedfordshire. Progress in engineering of *Chlorella* has been slowed by the lack of reliable transformation protocols. In this study transformation protocols based on electroporation and biolistics were investigated with the goal of reliably transforming Algenuity’s proprietary *C. vulgaris* strain. The literature surrounding these transformation methods was systematically reviewed and informed the protocol development process. RNAseq analysis of the closely related species *Chlorella variabilis* informed the design and assembly of a Golden Gate (GG) based *Chlorella* MoClo toolkit including promoters, terminators and antibiotic resistance genes. Regulatory elements from a *Chlorella* virus and *Chlamydomonas reinhardtii* were also included in the toolkit. From these parts antibiotic resistance cassettes were assembled and used in trials to develop an electroporation or biolistic transformation protocol for *C. vulgaris*. Attempts to transform the proprietary strain by these means were unsuccessful but a transformation technique based on *Escherichia coli* conjugation was successfully employed enabling the characterisation of the parts assembled. Results of conjugative transformation suggest that use of the *C. reinhardtii* RbcS2 intron1 in antibiotic resistance CDS reduces transformation efficiency and that use of the *C. variabilis EF1* promoter drives better expression than the HSP70-RbcS2 fusion promoter from *C. reinhardtii*. The molecular toolbox of the marine diatom *P. tricornutum* is more advanced than for *C. vulgaris* but lacks strong, inducible promoters. As such the *AP1* promoter (P*AP1*), upregulated under conditions of phosphate stress, and the METE promoter (P*METE*), upregulated in the absence of vitamin B12 (B12), were investigated accordingly to bring the engineering potential of *P. tricornutum* inline with other synthetic biology (SynBio) chassis. Reporter constructs were assembled with the promoters of interest cloned upstream of a Venus fluorescent protein and used to transform *P. tricornutum*. Initial characterisations of P*AP1*::Venus lines suggested that it suffered from leaky expression. Incorporation of the native terminator or intron did not alleviate the leakiness. However, the P*AP1* was successfully switched off when high concentrations of phosphate were supplied in the media. The P*METE* was shown to drive strong expression and can be modulated by varying the concentration of B12 in the growth media. The P*METE* was characterised relative to the P*EfTu* and the widely used P*LHCF1* and shown to drive stronger expression. Transformants harbouring a lethal expression cassette encoding barnase under the regulation of the P*METE* were obtained indicating that the P*METE* could be effectively turned off. These lines were unable to grow when subcultured in B12 deplete media. Including the METE terminator (T*METE*) in reporter constructs drove 2 fold higher Venus expression than was observed when using the standard *LHCF3* terminator (T*LHCF3*). Truncating the P*METE* resulted in decreased expression but no loss of B12 regulation. The P*METE* was most strongly expressed during exponential phase with expression dropping off through stationary phase. By simulating semi-continuous culturing, it was possible to maintain cells in log phase and as a result high activity of the P*METE*. To demonstrate the industrial relevance of this work the P*METE* was used to regulate heterologous production of the diterpene casbene. Casbene synthase (CBS) catalyses the conversion of geranylgeranyl pyrophosphate (GGPP) to casbene and represents the first step in the synthesis of medicinally important derivatives such as lathyranes and jatropholanes. Transgenic lines harbouring an expression cassette containing CBS from *Jatropha curcas* under the the regulation of the P*METE* produced casbene at titres of 100 µg/l (10 fg/cell). Higher casbene titres were observed, 750 µg/l (20 fg/cell), when lines were grown in F/2 supplemented with 10x nitrate, phosphate and trace elements. Varying the concentration of B12 in the media enabled tuneable casbene production with titres ranging from 0 to 180 µg/l when 1 µg/l or no was included in the media. Tagging CBS with Venus facilitated the identification of higher casbene producing transformants with multiple lines identified that produced approximately 2 mg/l. In contrast to Venus fluorescence assays casbene production was not improved by substituting the standard T*LHCF3* with the T*METE*.
  • ItemOpen Access
    Logging impacts the ecology of molecules in headwater streams
    Freeman, Erika
    Forest management and harvest offer a promising means of combating climate change by removing CO2 from the atmosphere. However, most forest carbon (C) is held in soils. Thus, by disturbing soils and altering hydrology, forest management and harvest potentially displace large amounts of C from forest soils into aquatic ecosystems. My dissertation seeks to understand the fate of this forest C as dissolved organic matter (DOM) into various aquatic endpoints by tracing its molecular composition along the ephemeral water film that begins in upland soils and ends in streams. The fate and function of DOM in aquatic ecosystems are strongly affected by its chemical properties. Thus, recent progress in the molecular characterization of DOM has opened a new line of inquiry into harvest impacts on aquatic ecosystem functioning. My thesis advances this line of inquiry by applying high-resolution mass spectrometry to study the effect of forest disturbance on DOM in soils and connected streams. Chapter 1 of this thesis gives a general overview of the molecular nature of terrestrial DOM sources, how these sources may be altered by harvest, and the subsequent transfer, fate, and properties of DOM once in streams. It also outlines the specific objectives I address with a combination of field experiments and synoptic surveys in the Batchewana watershed in Ontario, Canada. In Chapter 2, I tracked DOM along soil depths, and hillslope positions in four replicate forest headwater catchments of the Canadian hardwood forest. I related DOM composition to soil microbiomes and physical chemistry to establish baseline conditions before a harvest experiment. I found that DOM changed similarly along soil-aquatic gradients, irrespective of differences in environmental conditions. My results implicated continuous microbial reworking that shifts DOM towards a shared pool of compounds in soils. Such general degradation patterns can inform the management of soil-to-stream carbon losses by predicting DOM composition and its downstream reactivity along environmental gradients. In Chapter 3, I quantified and characterised the effects of logging on DOM composition over three years using the four experimental catchments from Chapter 2. Two catchments were experimentally logged, while the remaining two were left as controls. I found that DOM concentration in stream water from logged catchments increased in a pulse during the first year, but only the changes in the quality of DOM persisted. Using ultrahigh-resolution mass spectrometry, I showed that DOM released from deforested catchments was energy-rich and more chemically diverse, likely because of higher hydrological connectivity with intermediate and deep soil layers. I estimated that while logging increased the overall annual flux of dissolved organic carbon by approximately 8.5% of the extracted wood carbon, the exposure of deeper soil through logging released previously stable soil organic carbon to streams. The resulting changes to the molecular composition of DOM within headwater streams persisted for at least two years after logging, potentially disrupting aquatic ecosystems and making streams more likely to release terrestrial C into the atmosphere. In Chapter 4, I examined the chemical properties of DOM in stream water from over 200 Canadian headwater streams in an area with historical forest harvest. I demonstrated that using the fluorescence properties of streams, the effect of harvest, although detectable on a large spatial scale, is relatively minor compared to the effects of forest types and wetness gradients. These results have implications for land-water linkages under a changing climate that shift terrestrial sources of DOM. Finally, in Chapter 5, I discuss the implications of my findings for understanding the coupling between terrestrial and aquatic ecosystems and propose avenues for future research.
  • ItemOpen Access
    Exploring B vitamin biosynthesis and exchange in understudied marine stramenopiles and their communities
    Absolon, Dominic
    Microbes are the dominant form of life on Earth. Environmental sequencing has revealed the extent of the diversity of microbial life in all environments, not least the ocean. In this vast aquatic environment microbes, both prokaryotic and eukaryotic, play essential roles in the cycling of nutrients, primary productivity and form the basis of all food webs. These single-celled organisms do not live in isolation, rather they form complex communities, a dominant feature of which is the exchange of nutrients. In many areas of the ocean the concentration of some macro and micronutrients are limiting to the growth of some organisms. In addition, many organisms appear to have dispensed with the ability to biosynthesise various micronutrients, for example, B vitamins. Instead, they rely on an external source to satisfy their requirement. Here I explore the role of B vitamin exchange in microbial communities, with a particular focus on the marine protists of the stramenopile group of eukaryotes. An initial investigation took environmental sea water samples and applied different B vitamin and macronutrient amendments to assess if any of these compounds were limiting for the growth of the microbial community as a whole in this area of the ocean (English Channel), or if growth was co-limited by both B vitamin and macronutrient concentration. The results suggested that none of the B vitamins tested were a limiting factor for growth of the majority of species, either prokaryotic or eukaryotic. However, some changes to the microbial assemblage were observed when assessing the relative transcriptional activities of the organisms in the samples. For example, the bacterial genus Litorivivens was more transcriptionally active (high ratio of ribosomal RNA reads to ribosomal DNA reads) under the addition of vitamins B2, B3, B5, B6 and B9. The eukaryotic genera Ostreococcus and Picochlorum were also more active in the same condition. The stramenopiles display considerable variety in morphology and lifestyle, from parasitic soil-based oomycetes to photosynthetic planktonic species such as the diatoms. A comparative genomics approach was employed to investigate the B vitamin biosynthetic capabilities of this diverse group and therefore deduce any likely auxotrophies for these nutrients. Identifying auxotrophic species hints at potential requirements for nutrient exchange with an external partner. The results of this study showed that the metabolism of stramenopile species is as diverse as the physical attributes of the members of the group. Of the species analysed in this experiment 64% were predicted to be auxotrophic for B12 and this trait was spread across the group in a mosaic fashion. For the vitamin B3 the analysis shows a clear dichotomy in the utilisation of one of two alternative pathway branches. This indicates multiple gene transfer events have occurred across the group to allow for this change in metabolic routes. Biosynthesis for some of the B vitamins, namely B5 and B6 appears to be a universal trait in the stramenopiles. The results also demonstrate that B vitamin auxotrophy, in general, is more common in the heterotrophic species of the group than in the phototrophs. Finally, metagenomics was employed to probe the community surrounding an enigmatic marine stramenopile, Incisomonas marina, one of only a few MAST (MArine STramenopile) species that have been cultured. MASTs represent the hitherto understudied and unknown heterotrophic cohort of the stramenopiles. This is in contrast to the comparatively well studied and understood ochrophytes, the photosynthetic members of the stramenopiles, who have thousands of representatives in culture. I. marina was originally isolated from Nova Scotia, Canada with a cohort of bacterial species, with which it has been maintained in culture. This provides a closed community to interrogate for nutrient exchange and interaction. Analysis of pathways for B vitamin and amino acid biosynthesis revealed that no one single species of the one eukaryote and 23 bacterial species in the community encodes a complete biosynthesis pathway for the full suite of either the B vitamins or the amino acids. This suggests some level of exchange for these essential nutrients indicating a community-based approach to metabolism. Overall, the work outlined in this thesis suggests that B vitamins play an essential role in shaping the communities within which stramenopiles reside. Given the ecological prevalence of this group, these types of interactions between members of the community to share nutrients and exchange metabolites are essential to the health and function of the environments in which they live. Furthering our understanding of these processes will aid our understanding of microbial communities as a whole and the biogeochemical processes that they dictate.
  • ItemOpen Access
    Characterising natural genetic variation in dynamic photosynthesis and photoprotection in sorghum
    Vath, Richard
    Non-photochemical quenching (NPQ) photoprotective processes are key for protecting photosynthetic machinery from excess light energy. Improving the regulation of NPQ in leaves during dynamic light conditions has been identified as a potential route toward increasing photosynthetic efficiency in the world’s most important crops. Prior studies have revealed intraspecific variability in other photosynthetic efficiency-related traits, but variation in photoprotective capacity within crop species, particularly those utilizing the C4 photosynthetic pathway, is not yet well-characterized. The aim of this research project was to determine the genetic underpinnings of photoprotective traits in *Sorghum bicolor* and improve understanding of the balance between photosynthesis and photoprotection in dynamic light conditions. Utilising a high-throughput chlorophyll fluorescence technique, a panel of 869 field-grown sorghum accessions was screened for multiple traits related to NPQ kinetics over two growing seasons. An ensemble approach combining genome and transcriptome-wide association studies was used to characterise the genetic architecture of NPQ in sorghum, and several high-confidence loci correlated with the observed variation were identified. These results were validated via re-screening of NPQ kinetics of selected sorghum accessions displaying contrasting genotypes at two of these loci. Sorghum’s photoprotective response was also compared and contrasted with that of three other C4 crops and one C4 model species, in order to contextualise photoprotection in sorghum within a broader C4 photosynthetic phylogeny. Subsequently, two sorghum accessions with strongly contrasting NPQ phenotypes were used to investigate the effect of photoinhibition on the coordination between the C4 carbon concentrating mechanism and Calvin-Benson-Bassham cycle CO2 fixation. Imbalances between these cycles during stress conditions likely represent a loss of photosynthetic efficiency in the field, potentially resulting in decreased productivity, but there presently exists minimal knowledge of the effect of photoinhibition on coordination of mesophyll/bundle-sheath cell carboxylation activity. A combined carbon isotope/leaf-level gas exchange system was developed and utilised for real-time measurement of carbon isotope discrimination during photosynthesis in high-intensity steady-state and fluctuating light conditions, to investigate the effects of high-light treatments on bundle sheath leakiness– an indicator of loss-of coordination between cell types. The results suggest that the C4 photosynthetic apparatus is robust under both fluctuating and steady-state high light conditions and that NPQ capacity may partially explain susceptibility to photoinhibition. This project has identified genetic loci underlying key photoprotective traits in sorghum and improved understanding of the interplay of photosynthesis and photoprotection under dynamic light conditions. The resulting knowledge of the genetic basis and physiological implications of variation in photoprotection will help guide crop improvement via both traditional breeding and biotechnology based approaches.
  • ItemEmbargo
    Convergent evolution of betalains in Caryophyllales
    Walker-Hale, Nathanael; Walker-Hale, Nathanael [0000-0003-1105-5069]
    In this thesis, I investigate the evolution of betalains in Caryophyllales. Betalains are a group of red and yellow pigments, which in plants occur only in the order Caryophyllales. Here, betalains replace the otherwise ubiquitous anthocyanin pigments, and the two pigment types appear mutually exclusive. Some lineages of Caryophyllales maintain anthocyanins, and the distribution of the two pigments with respect to relationships within the order suggests a complex evolutionary history, that allows for the possibility of repeated convergent shifts from anthocyanin to betalain pigmentation. First, I begin by framing my thesis with a review of the concepts of convergence and parallelism, including the connection between these terms and concepts of adaptation and constraint. I then discuss convergence at the level of molecular sequence data, and explore ideas of convergence at multiple hierarchies and their integration in the evolution of complex traits. Second, I conduct a reanalysis of the evolution of betalains, using the latest phylogenomic hypothesis of Caryophyllales relationships and up to date pigmentation data. I show that these data support up to four origins of betalains. I re-examine the phylogeny of DODA, a key gene in the betalain biosynthesis pathway, and show that the distribution of betalain-related enzyme activity is consistent with multiple origins of high activity following repeated gene duplications, consistent with inferred origins of betalains. I argue that multiple transitions to high activity in this enzyme have facilitated multiple origins of betalain pigmentation. Third, given this scenario, I assess the molecular signal for convergent evolution in DODA lineage. I reconstruct ancestral sequences and show that experimental testing of their activity supports multiple origins of high activity. I infer historical substitutions and show that a complex history of convergent molecular evolution has occurred, across multiple branches of the gene tree and in different order depending on origin. I support my findings with a suite of statistical methods and discuss how these approaches differ and how they can be used to interrogate betalain evolutionary history. Fourth, I explore convergence at the level of genome structure. I sequence and assemble three new Caryophyllales genomes with long reads. Two key genes of the betalain pathway are clustered in the Beta vulgaris genome, and I use comparative genomics to explore the evolutionary history of this clustering. Strikingly, I show that a pattern dispersed synteny of betalain-implicated DODA paralogues, has occurred multiple times via different mechanisms. I discuss the potential significance of this finding with respect to syntenic evolution and the evolution of betalains. Finally, I use comparative expression to explore betalain origins. I design pigment induction experiments in diverse taxa representing multiple origins and measure associated gene expression with RNA-seq. I interrogate these data with coexpression techniques and provide new insights into comparative gene networks and betalain regulation.
  • ItemOpen Access
    A comparative analysis of C3 and C4 photosynthesis under dynamic light conditions
    Arce Cubas, Lucía
    By 2050, human population growth is predicted to require to a 100-110% rise in global food production, and since traditional targets for crop advancement are falling behind rising demand, improving photosynthesis to increase crop yield has become a major global effort. The response of plants to changes in light intensity has been identified as a source of inefficiency for photosynthesis, as in the dynamic environments of the open field, where crop canopies are subject to constantly changing light intensity, lags in photosynthetic responses can amount to up to a 40% loss in daily carbon assimilation. Although remarkable progress has been made, the vast majority of the work on dynamic light photosynthesis has been conducted in C3 species, despite the undeniable global importance of C4 crops to global food supplies. The benefits of the C4 carbon concentrating mechanism under steady state conditions are well-established, but it is less clear how the C4 pathway affects the dynamic light response. This thesis employs a comparison between phylogenetically linked C3 and C4 species across three different genera to compare C3 and C4 photosynthesis under non-steady state conditions. The findings presented in this thesis enhance our understanding of the effects of the biochemical and anatomical features of the C4 pathway on photosynthetic responses to dynamic light, and on the potential impact of specific engineering strategies for the improvement of photosynthesis in C4 species.
  • ItemOpen Access
    Understanding Meiotic Recombination and Genomic Organisation of Plant Species
    Wlodzimierz, Piotr; Wlodzimierz, Piotr [0000-0003-1040-7878]
    Reciprocal exchange of eukaryotic genetic material during meiotic crossing over is a major source of genetic variation in sexually reproducing species. Crossover events are not distributed randomly along chromosomes and some regions of the genome, like the centromeres, rarely undergo recombination. Modifying crossover levels and distributions via genetic engineering may provide effective tools for plant breeders to accelerate strain improvement. Despite low recombination rates and their evolutionary conserved function as kinetochore assembly loci, centromeres exhibit some of the highest levels of variation within eukaryotic genomes. Discovery of centromere structure has been hindered by the challenging process of genomic assembly of repetitive regions, as many species contain megabase-long arrays of centromeric tandem repeats. While new long-read DNA sequencing technologies allow for more accurate assembly across the centromeres, methods for their annotation are also required. In this thesis, I present the development of Tandem Repeat Annotation and Structural Hierarchy (TRASH) software that facilitates analysis of tandem arrays, including centromeric satellite arrays, without prior knowledge of repeat families present in an assembly. I benchmarked TRASH against other software and found it to advance on the current annotation and analysis methods. I used TRASH to analyse in depth the centromeric structures of multiple accessions of metacentric *Arabidopsis thaliana*, *Arabidopsis lyrata*, *Brassica oleracea* and holocentric *Rhynchospora* genus species. I also present progress towards investigation of the HEI10 meiotic E3 ligase and its role in modulation of crossover levels. Specifically, quantification of the dosage effect of *HEI10* multi-copy lines of tomato and *Arabidopsis* on the crossover recombination landscape. Together this work contributes to a better understanding of plant centromeric regions and meiotic recombination modulation. It also provides a novel bioinformatics tool for centromere sequence analysis and tandem repeat identification to the scientific community,
  • ItemOpen Access
    Propagation of engineered cellular properties to colony scales in bacterial populations
    Kan, Anton
    Biological development is a complex and elegant process that generates the vast array of form and shape found in nature. This process is an example of self-organization, whereby a global pattern of cells is generated by the propagation of local intercellular interactions to larger scales. Understanding this process is one of the fundamental challenges in biology and presents an opportunity to engineer the organization of biological matter. The work described in this thesis has developed a simplified bacterial system to study biological self-organization, focussing on quantifying the influence of physical cellular properties and interactions on global cellular arrangements within colonies. The approach of this project was to use Escherichia coli bacteria, building a range of synthetic genetic tools to generate and regulate cellular properties and interactions, aiming to regulate colony organization. This approach of regulating control over cellular properties within a simplified framework has significant advantages in probing biological self-organization, avoiding the complexity present in natural multicellular systems. A range of microscopy techniques and image processing tools were established to measure cellular properties and cellular arrangements, as well as the dynamics of colony growth. The experimental work was closely integrated with biophysical computational cellular models using the CellModeller multicellular modelling software. In order to regulate cellular organization within colonies, the first objective was to understand the native organization in E. coli colonies. For this, segmentation was performed on whole colonies, as well as time-lapse microscopy of growing colonies to find the spatial pattern of growth. The computational model was informed using this experimental data, setting the properties of single cells, then used in combination with the data to understand the processes driving global cellular arrangements. The results showed that bacterial colonies contain multiple domains of locally aligned cells, with domain size dependent on cell length. As aligned domains grow, they buckle and break alignment, leading to the development of fractal-like boundaries between lineages of cells. As a colony grows larger, the central region ceases growth, creating a velocity gradient that aligns cells with the radial direction of the colony. The experimentally observed cellular arrangements were also generated in the computational model through the designation of only single cell parameters, indicating the order within colonies was generated by physical interactions. A toolbox of synthetic genetic mechanisms was constructed to alter cellular and colony properties, alongside computational models of the mechanisms. Genes were used to alter cell shape, resulting in spherical cells of various sizes, as well rod shaped and filamentous cells. Intercellular adhesive interactions were generated through the expression of an adhesin, which generated altered microcolony morphology. Furthermore, synthetic symmetry breaking mechanisms were created to generate distinct domains of gene expression in colonies arising from single cells. Spatial patterns were generated through a system of segregating incompatible plasmids, and temporal patterns of gene expression with a quorum sensing circuit. Two of the engineered mechanisms were combined to investigate the structural effects of intercellular adhesion. This was done by combining characterized mechanisms for intercellular adhesion and spatial patterning, generating colonies with distinct spatial domains with varying adhesive strengths. Quantification of the morphology of the spatial domains found that adhesion elongated the fractal-like boundary between domains, but only when both domains were adhesive. Modelling further indicated the physical origin of this result, finding the same result in simulated colonies. Time-lapse microscopy of bacterial colonies found that adhesive interactions increased rotational motion during growth, which elongated the boundaries between domains, expanding the area of interaction. The work described in this thesis has demonstrated that physical interactions can have significant impact on bacterial self-organization, and provides a platform to study and regulate such processes. The work has shown that even relatively simple organisms collectively display a remarkable amount of order, generated by physical processes. Furthermore, the results display a close correspondence of computational models to experimental data, providing a computational platform to study and design self-organizing processes in bacterial systems, which can subsequently be built in vivo. Given that the properties of individual cells are relatively simple to engineer, understanding their propagation to larger scales can radically simplify the engineering of macroscopic biological structure.
  • ItemOpen Access
    Systems analysis of Crassulacean acid metabolism (CAM) physiology and molecular biology
    Chomthong, Methawi
    The main theme of this thesis was to explore the regulatory landscape of CAM using systems biology approaches. The scope of the regulatory landscape was drawn around the mesophyll metabolism of the dicotyledonous obligate CAM genus, *Kalanchoe*, even though the computational frameworks that had been developed here can also be applied to other species. The first result chapter presented the Ordinary Differential Equation modelling of Crassulacean acid metabolism (Chapter 2) which was developed to capture the classical gas-exchange patterns as well as the responses to perturbation conditions. The findings suggested that the model was sufficient to explain the classical gas-exchange pattern whilst was also responsive to the perturbations. Nonetheless, the model parameters which represented the protein activities indirectly captured the upstream regulatory controls. Thus, the following result chapter shifted the focus to explore a more upstream level of regulations at the level of gene expression. The second result chapter presented the Gene Regulatory Network Inference of *Kalanchoe fedtschenkoi* (Chapter 3). This chapter identified potential transcriptional regulators on different functional compartments of CAM including the following: Carboxylation subnetwork, Decarboxylation subnetwork, Circadian subnetwork and Stomatal subnetwork. This chapter highlighted the potential transcriptional regulators of key CAM genes, for example, PEPCarboxylase (PEPC), PEPCkinase (PPCK), pyruvate orthophosphate dikinase (PPDK), and pyruvate orthophosphate dikinase regulatory protein (PPDK-RP). Overall, the Gene Regulatory Network Inference provided the ranking of the potential transcriptional regulatory candidates on CAM genes. Hence, a reasonable step forward would be to probe for direct binding evidence through molecular approaches. The first step towards accessing the chromatin landscape with ATAC-sequencing technique was the Nuclei isolation followed by the flow cytometry separation technique for *Kalanchoe fedtschenkoi* which was presented as the final result chapter (Chapter 4). To conclude, this thesis showed that the minimal mechanistic model at the level of protein functions can capture CAM gas-exchange patterns under various scenarios. Subsequently, a more upstream level of regulatory controls was explored across the genome with the Gene Regulatory Network Inference method. The key findings highlighted the potential transcriptional regulations of key CAM genes in addition to the regulations at the level of protein activities. Finally, the nuclei isolation was conducted as an initial step for a future molecular experiment to probe for chromatin accessibility for the CAM model species.
  • ItemOpen Access
    Molecular Physiology of the Chlamydomonas Pyrenoid
    Caspari, Oliver Dominik
    Operation of the CO2-concentrating mechanism (CCM) in the model green alga *Chlamydomonas reinhardtii* relies on confining Rubisco in a proteinaceous microcompartment, the chloroplastic pyrenoid. Despite a long history of research, this enigmatic structure lacks a full definition both in terms of physiological function as well as molecular composition. The work presented here used a unique set of pyrenoid-less mutants (which differ from wild-type only with respect to the genes coding for the small subunit of Rubisco) to address two key questions: (i) the function of the pyrenoid in photosynthesis in enabling the CCM to supply Rubisco with inorganic carbon under CO2 limiting conditions; (ii) the mechanism of Rubisco aggregation underlying pyrenoid formation. Firstly, cells that are unable to aggregate Rubisco were found severely limited by access to CO2, yet fully able to compensate any structural changes within the chloroplast. Secondly, *in silico* and *in vitro* analyses of Rubisco protein interactions established that a linking agent is required for Rubisco aggregation. A Rubisco interactome was characterised using native gel electrophoresis and co-IP assays followed by mass spectrometry. In a complementary approach, a forward genetic screen based on high-throughput immunofluorescence localization of Rubisco aimed to identify key pyrenoid assembly factors. The present study combined and developed a wide range of molecular, physiological and computational techniques, to show that Rubisco aggregation forming the pyrenoid is achieved through a complex network of protein interactions in order to effectively supply CO2 via the CCM.
  • ItemEmbargo
    The Role of Arabidopsis thaliana Cyclic Nucleotide-Gated Channels 2, 6, and 19 in Extracellular ATP Signalling
    Ning, Youzheng
    Calcium (Ca2+) is known as a secondary messenger in plant growth, development, and stress perception. Plants generate stress-specific Ca2+ signals (Ca2+ signatures) to translate extracellular signals into cellular responses. Extracellular Adenosine 5’-triphosphate (eATP) is a crucial signalling molecule in plants but poorly understood. Upon abiotic and biotic stresses, ATP is released into the extracellular matrix, where it is referred as eATP, to regulate plant growth and development, salt tolerance, immunity, and the Damage-Associated Molecular Patterns (DAMP) response. Two plasma membrane (PM) eATP receptors DOES NOT RESPOND TO NUCLEOTIDES1 (DORN1/P2K1) and DORN2/P2K2 have been identified in Arabidopsis thaliana in the past decade. The perception of eATP leads to downstream changes of secondary messengers, including cytosolic free calcium ([Ca2+]cyt) increase, reactive oxygen species (ROS) and nitric oxide (NO) production. Subsequently, [Ca2+]cyt regulates production of defence-related hormones (such as jasmonic acid, JA), plant elicitor peptides (PEPs), and indolic glucosinolates (IG). However, the PM calcium channels underpinning eATP-induced [Ca2+]cyt increase remain poorly studied. Here, my PhD project revealed the role of Cyclic Nucleotide-Gated Channels (CNGC) 2, 6 and 19 as putative calcium channels in eATP signalling. By employing (apo)aequorin as a Ca2+ reporter, CNGC2 was found to be involved in Arabidopsis root’s [Ca2+]cyt responses to both eATP and eADP. Transcriptional analysis showed that the expression of CNGC6 and CNGC19 was upregulated by eATP in a DORN1/P2K1- and CNGC2-dependent manner. The involvement of CNGC6 and CNGC19 in the root’s eATP-induced [Ca2+]cyt increase was then discovered and (for CNGC19) investigated further using GCaMP3 as a [Ca2+]cyt reporter, as a function of phosphate nutrition. Notably, both CNGC2 and CNGC19 were suggested to modulate the root’s transcription of components of eATP-activated defence pathways. As a result, loss-of-function mutants of CNGC19 exhibited increased susceptibility to infection by the parasitic cyst nematode, Heterodera schachtii.