School of BioSciences - Theses
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ItemGenomically informed gene drive modelling( 2022)CRISPR/Cas gene drives are a focus of genetic biocontrol for pest species. They have the potential to radically affect pest species, by making them more manageable or by eradicating them. However, it is not yet fully understood how the elements of a gene drive interact to guide the progression of a gene drive. We explored how we can design gene drives that are safer, either by being temporally limiting or spatially limiting, through a modelling framework. Our modelling included a range of variables, with the addition of genomic information to infer the homing efficiency of the gene drive. We showed that there was no single variable that differentiated between the outcomes of a gene drive. Granted some variables were more influential in determining the outcome than others. The degree of dominance of the selection coefficient was shown to be strongly influential on the equilibrium outcome. While the interaction between conversion efficiency and resistance was shown to strongly influence the Temporary outcome. Furthermore, we showed that internal dynamics of a gene drive can be regulated by the variables of the gene drive. This provided insight into where effort should be directed in gene drive design to achieve the intended outcome of a gene drive, as well as controlling the progression to that outcome. The inclusion of genomic data in CRISPR gene drive modelling allowed for localisation of the gene drive due to genetic variation alone. Finding loci in the genome where there were allele frequencies differences allowed us to model gene drives that were highly efficient in the target population and poorly efficient in off-target populations. This conversion efficiency differential allowed for sustained gene drive localisation in spite of migration and selection. Population suppression was explored in our modelling to better understand how we could create sustained localised suppression. We showed sustained population suppression was possible through incomplete distortion of the sex ratio of the progeny. A deterministic gene drive model was developed to solve for equilibrium points for a range of migration rates and selection coefficients. These equilibria can be used as thresholds for gene drive design and monitoring. This work aims to further develop our understanding of how gene drives are likely to progress when released. We focussed on characterising which aspects of a gene drive were most important in determining both their progression and outcome. The inclusion of genetic information in our modelling revealed a new avenue that can be exploited to achieve gene drive localisation. This modelling work will aid in the design process of gene drives to increase our confidence that gene drives will work as intended.
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ItemExploring the cancer transcriptome with novel bioinformatics approaches( 2022)Currently three out of every 10 deaths within Australia will be a direct consequence of cancer. Cancer is a complex and genetically heterogeneous disease that is, as a consequence, effectively unique to each individual. However, there are common driving events, phenotypes, and risks that can segregate cancer within tumour types and subtypes. These groupings are beneficial as they can both inform treatment regimes and yield new targets for pharmaceutical development. Next Generation Sequencing (NGS) of RNA has enabled measurement of the abundance and makeup of a sample’s transcriptome, which through bioinformatics analysis, can reveal the rich interplay between genetic mutations and their functional and phenotypic consequences. This thesis focuses on three key transcriptome projects. The first project developed the ALLSorts software which is the first publicly available and open-source classifier for determining subtypes of B-Cell Acute Lymphoblastic Leukemia (B-ALL). The purpose of this tool is to provide researchers with an accurate method for using transcriptome data to quickly label B-ALL samples according to 18 subtypes. Subtyping is becoming part of clinical standard-of-care, informing targeted pharmaceutical treatment and/or treatment intensity. The second project, Slinker, is a publicly available and open source visualisation tool that can be applied to any gene that highlights splicing variation between a case and controls. Novel splicing is regularly observed across a variety of diseases, including cancer, and can lead to a significant alteration of the final transcript, possibly transforming it into a pathogenic driver. Slinker is novel in that it utilises the superTranscritome method to create succinct visualisations by removing redundant features. The final project in this thesis is an analysis of the utility of long read transcripts as a transcriptomic reference, specifically within a spatial context. Three references were compared: the hg38 reference transcriptome, the long reads themselves as a reference, and both combined. Each had gene expression quantified through highly accurate, short read technology. The combined reference resulted in both a higher mapping rate and novel expressed sequences, of which one belongs to a gene that is a known prognostic marker for the oropharyngeal head and neck cancers that this method was applied to.
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ItemDeciphering the molecular mechanisms underpinning oil biosynthesis in Salvia hispanica L. (Chia)( 2022)Salvia hispanica L. (chia) is an oil seed plant rich in omega-3 polyunsaturated fatty acids, proteins, and antioxidants. Daily consumption of chia seeds has been associated with health benefits related to cardiovascular system and cognitive function. The link observed between the nutraceutical composition of chia seeds and the health benefits associated with it prompted extensive research into the contents of chia seeds. The study of oil biosynthesis pathways in the model organism Arabidopsis thaliana as well as other oil seed plants has been an active research area aiming to unravel the mechanisms of oil production. However, the underlying genetic mechanisms that regulate oil biosynthesis, particularly in non-model organisms such as S. hispanica, are little understood. One of the several biological roles assigned to plant lipids is their involvement in abiotic stress responses. The cause-effect relationship of oil biosynthesis and stress response in plants becomes particularly important under extreme environmental conditions. The rise in global temperature is thus threatening the yield and quality of S. hispanica’s seed oil quality. The genetic make-up of S. hispanica was unstudied prior to this PhD study, thereby hindering the discovery of genes and regulatory elements that determine its unique traits. Advances in high-throughput genomic technologies including Illumina short-read sequencing, Oxford Nanopore Technology long-read sequencing, and Hi-C chromosome conformation capture technique enabled the creation of a high-quality near-complete chromosome-level reference genome for S. hispanica. The evolutionary study of the S. hispanica genome through comparative genomics revealed novel aspects related to the omega-3 fatty acid accumulation in chia seeds. The hypothesis that due to a recent whole genome duplication highly expressed genes regulate oil biosynthesis was rejected. Instead, the evolutionary expansion of the stearoyl-ACP desaturase (SAD) gene family due to tandem duplications was identified as the key factor of efficient oil biosynthesis in S. hispanica. Along with global warming, heat waves in the Kimberly region of Australia are anticipated, their effect is a major concern for chia growers. In this thesis, an integrated transcriptomic (RNAseq) and lipidomic (LC-MS) approach was used to demonstrate an effective basal thermotolerance in S. hispanica in response to heat shock and prolonged heat stress. The successful recovery of lipids and transcripts in S. hispanica leaves that undergone heat stress is proposed to be associated with Ca2+ signalling and cytosolic transport pathways as well as two distinct membrane lipid-remodelling mechanisms. An increase in the abundance of unsaturated lipids, dominated by the triacylglycerol (TG) family, is proposed to cause stabilisation of membrane fluidity. In parallel, an analysis of differentially expressed genes indicated that the phospholipid diacylglycerol acyltransferase (PDAT) and the diacylglycerol O-acyltransferase (DGAT) genes play a pivotal role in heat induced biosynthesis of TGs in the endoplasmic reticulum and detoxifying the chloroplast from free fatty acids. The reference genome of S. hispanica generated as part of this PhD study will greatly assist the plant science and plant breeding communities to study the molecular mechanisms of S. hispanica in the future. Features of the generated high quality Hi-C contact map such as chromosomal territories and regulatory interaction of enhancers and promoters will provide new insight into the molecular genetics underlying the unique traits of S. hispanica. This dissertation further contributes to a better understanding of fatty acid biosynthesis in S. hispanica and the role of lipids in response to heat stress in general. Together the results of this PhD will aid the development of novel agronomical crops with improved seed oil content or enhanced resistance to abiotic stresses while mitigating the detrimental impacts of climate change.
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ItemDissecting the contribution of structural gene and regulatory variation in metabolic resistance to insecticides in Drosophila melanogaster( 2022)Strong selection pressure imposed by insecticide usage has allowed resistance to evolve and spread in insect populations. One mechanism underlying resistance, increased insecticide metabolism, is often linked to increased expression or activity of enzymes belonging to four major families: cytochrome P450s, esterases, glutathione S-transferases and uridine diphosphate-glycosyltransferases. There is a growing body of evidence that the capacity for metabolic enzymes to confer insecticide resistance is a by-product of their evolved capacity to metabolise xenobiotics present in the natural environment. This has led to the hypothesis that insect populations may contain an array of metabolic enzymes that can potentially provide resistance to insecticides, despite not being optimised for insecticide metabolism in terms of their expression or structure. This raises questions about the genetic changes required for metabolic resistance to evolve and the number of enzymes in a given species that have the potential to confer resistance. This study addresses these questions by exploiting two well-defined model systems – the resistance genes Cyp6g1 in Drosophila melanogaster and LcaE7 in Lucilia cuprina. A multi-faceted approach using in vivo, in vitro, and in silico techniques has been deployed to explore these questions. The first aim of this study was to evaluate the resistance potential of five D. melanogaster P450 genes closely related to Cyp6g1. This was achieved through transgenic overexpression regulated by the Accord promoter responsible for elevated levels of Cyp6g1 expression in natural populations of D. melanogaster. Homology models were also created for all six of these P450s. Of these genes, only Cyp6g1 and Cyp6g2 were able to confer resistance towards the insecticides tested - nicotine and the neonicotinoids, imidacloprid and nitenpyram. The second aim investigates the structure-function relationship of CYP6G1. Molecular docking alongside site-directed mutagenesis experiments were able to demonstrate that Phe123, Phe124, Thr219, and Val377 are involved in the metabolism of imidacloprid. Moreover, CYP6G1 variants with an increased capacity for imidacloprid metabolism were generated via Phe220Pro or Val308Ser replacements. However, only one of these mutations, Phe220Pro, confers increased levels of imidacloprid resistance. The third aim explores whether a highly efficient organophosphate hydrolase LcaE7 variant (R9) produced through laboratory-directed evolution could confer resistance when placed in an in vivo D. melanogaster system. Despite having very high levels of activity, this LcaE7 variant conferred lower than expected levels of organophosphate resistance. Activity-stability trade-offs in this evolved variant has reduced its capacity for organophosphate resistance, hence it is unlikely to arise and spread in natural populations of L. cuprina. The findings from this study further our understanding of the evolutionary options available for metabolic resistance and contribute to the capacity to predict the nature of resistance that may evolve, leading to better resistance management strategies.
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ItemQuaternary diversity dynamics of Australian reptiles( 2022)Predicting the outcomes of anthropogenic impacts on ecosystems is an essential step to counteract the recent biodiversity crisis. The Quaternary fossil record offers a unique opportunity to formulate such predictions by testing how ecological communities and / or species distributions change through time, e.g., in response to the repeated and intensifying shifts in global climate during the glacial-interglacial cycles. Such paleoecological information is particularly critical for ectothermic vertebrates, such as reptiles and amphibians collectively known as herpetofauna, as these groups comprise a high number of threatened species and are particularly sensitive to changing climates. Yet, in most cases, the investigation of long-term faunal dynamics requires a morphology-based taxonomic or ecological identification of fossilized elements. For herpetofauna this has been notoriously difficult, due to a lack of comparative knowledge about the osteological variation in modern taxa, underdeveloped osteological museum collections, and the prevalence of cryptic diversity. These difficulties pose a major challenge when paleontological data are intended to inform conservation, because applied conservation measures fundamentally rely on (species-level) taxonomy (e.g., the IUCN Red List). In this thesis, I test the recognizability of herpetofaunal species in the Quaternary Australian fossil record and apply alternative methods for inferring climate-related faunal dynamics, through a combination of quantitative paleontological and neontological methods. Australia is ideal for such an analysis as the continent comprises an exceptionally high herpetofaunal diversity as well as numerous Quaternary fossil sites, preserving a relatively continuous temporal sequence of reptile and amphibian fossils. I show in Chapter 1 that faunal change can be detected at higher taxonomic levels (above the species-level) and that changes in relative abundance of different reptile subfamilies over time correspond to changing aridity throughout a fossil deposit in western Victoria. This suggests that historical baselines for evaluating the stability of modern ecosystems may be established even in the absence of species-level taxonomic resolutions. The central aspect of this thesis is addressed in Chapters 2 and 3. Using a quantitative approach based on 3D geometric morphometrics, I leverage digital morphological data (CT scans) to test how reliable individual bones of agamids (Chapter 2) and varanids (Chapter 3) can be assigned to (modern) lower-level taxonomic or ecological categories. My results show that genus- or subgenus-level as well as ecological identifications can be confidently achieved in most cases (> 90%). Thus, these categories constitute appropriate groupings for the investigation of temporal diversity dynamics. In contrast, species-level identifications were generally less reliable and sensitive to incompleteness of the bones or sample size. These results add to the long-standing question of transferability of modern species boundaries to the fossil record and imply that a comparison of modern and past (species-level) biodiversity may be prone to identification errors, at least within these groups. Finally, in Chapter 4, I integrated fossil occurrences, generated through the quantitative identification framework developed in the previous chapters, with (paleo-)species-distribution modelling, population genomics and osteological data of modern specimens to examine the decline of the threatened Mountain Dragon (Rankinia diemensis). This integrative approach revealed a strong link between Quaternary climate change and ongoing habitat loss and fragmentation in this temperate-adapted agamid lizard. My results suggest that increasing temperatures will likely force R. diemensis to further shift its distribution to higher altitudes, leading to a reduction of suitable habitat and increasing fragmentation of populations as global warming proceeds. Overall, my thesis provides new insights into the possibilities and limitations of the Quaternary Australian herpetofaunal fossil record in a conservation-paleobiological context, as well as an extensive resource of virtual morphological data and a quantitative methodological framework for future research.
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ItemLight manipulation by Christmas beetles: quantification, mechanisms and ecological relevance( 2022)The brilliant and colourful appearances of Christmas beetles (Scarabeidae - Rutelinae) are famous for decorating eucalyptus trees during the Australian summer. Millions of years of evolution perfected the design of their hardened fore wings (elytra) to manipulate light and create striking optical effects. However, little is known about the exact underlying nanostructures or the ecological variables driving their evolution. Despite the recent increase in biophotonics studies, this remains the case for many organisms with striking colourful appearances. By studying light manipulation in Christmas beetles, I aim to develop methods and concepts generalisable to other organisms. Christmas beetles produce a wide variety of optical effects including saturated colours, black, and pearlescent white. Some species look mirror-like and metallic gold or brass. Moreover, these colours change with the viewing or illumination angle. Christmas beetles can also interact with long wavelengths outside of the human-visible spectrum, in the near-infrared (NIR), and reflect circularly polarised light, which makes them appear as different colours through the right and left lenses of 3D cinema glasses. To better understand this diversity, I proposed a method for its quantification, studied some of its underlying mechanisms and tested if it can be explained by ecological differences. To characterise the beetles’ optical effects, I proposed a generalization of existing spectroscopy methods and parameters to describe reflection profiles across the visible and NIR spectrum, regardless of their underlying mechanism. Ultimately these methods break down complex optical effects into simpler traits and can facilitate comparison between studies. The proposed terminology attempts to conceptually unify disparate fields (biology and optics) and allows a clear distinction with terms used to describe colour perception. To study the optical mechanisms in Christmas beetles, I analysed the architecture of their elytra. I discovered that unlike scarab beetles studied to date, three different Christmas beetle species use multicomponent photonic systems, with an upper layer acting as a green pigment-based filter and an underlaying broadband reflector, that particularly enhances NIR reflectance. For beetles with spiral nanostructures, I found a trade-off between polarisation and NIR reflectance. This diversity of photonic structures shows that beetles are a promising model for understanding complex optical properties of natural materials. To investigate ecological correlates of the optical effects in Christmas beetles, I used a biophysical essay and a phylogenetic comparative analysis. High reflectance reduced heating of the beetle elytra under controlled conditions, but I did not find any evidence that highly reflective species occur in hotter environments. Christmas beetles do not follow a simple eco-geographical pattern, possibly because their optical effects respond to species-specific combinations of environmental challenges. I demonstrated that diversity in optical effects and photonic mechanisms is more than meets the eye, even for a small group such as Christmas beetles. My thesis highlights the value of a cross-disciplinary approach where optical methods can spark stimulating biological questions and the comparative study of phylogenetically related species can inform species selection for photonics studies. The resulting conversation between the two disciplines accelerates progress in the search for the biological function of striking optical effects.
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ItemUnderstanding the role of companion of cellulose synthase1 (CC1) in maintaining cellulose synthesis under salt stress( 2022)Salt stress is one of the most detrimental abiotic stresses for plants, and substantially impacts plant biomass and agricultural productivity. In the last decades, revealing how plants cope with stress conditions and maintain growth under salt stress has been an important focus in plant research and agricultural development. The plant cell wall, which encases plant cells and functions as a cellular exoskeleton, is an important structure to cope with such stresses. One of the main components of the cell wall is cellulose, which is synthesized by cellulose synthase (CESA) complexes (CSC) at the plasma membrane by moving along underlying cortical microtubules. COMPANION OF CELLULOSE SYNTHASE (CC) 1 and 2 are components of the CSCs and links the CSCs to the microtubules. CC1 and CC2 function in maintaining cellulose synthesis under salt stress by supporting microtubules and CESA behaviors. However, the exact regulatory mechanisms of the CC1 and CC2 proteins remain largely unknown. In this thesis, the regulatory mechanisms of CC1 are investigated in more details. In Chapter 2, a comprehensive phylogenetic analysis shows that the CC protein family contains up to seven members in land plants, with six CCs present in the Arabidopsis thaliana (A. thaliana) genome. The chimeric constructs swapping the N-terminus of CC1 with different homologs, i.e., CC2-CC6, were generated and transformed into cc1cc2 double mutants. The phenotypic analyses showed that CC1 to CC4 and CC6 behaved similar to CC1 in supporting plant growth under salt stress, while CC5 did not. These inabilities of CC5 were due to its defects in microtubule-binding, microtubule bundling, and maintenance of microtubules stability under salt stress, as well as other changes in functionalities of the N-terminal part of the protein. CC5 was found to have a dominant negative effect on plant root growth. Furthermore, CC5 was specifically expressed in pollen and inhibited pollen germination under salt stress. These findings reveal functional differences among CC protein family members and improve our understanding of the four microtubule-binding motifs of CC1. In Chapter 3, an immunoprecipitation-mass spectrometry (IP-MS) analysis was performed to identify potential interactors of CC1 under normal conditions and salt stress. Among the candidates, FERONIA (FER) was selected for further study. It was found that FER interacted and phosphorylated CC1, and the phosphorylation of CC1 by FER negatively affected its in vitro microtubule-binding and microtubule-bundling abilities. In Chapter 4, in planta phospho-proteomic analysis was performed and potential phosphorylation sites of CC1 were identified. Here, BRASSINOSTEROID INSENSITIVE 2 (BIN2) was found to phosphorylate CC1, and the relevant phospho-null and phospho-mimetic mutants were generated and briefly characterized. These findings provide further insights into the regulatory mechanisms of CC1, which are important to better understand how plants maintain cellulose synthesis and growth under salt stress.
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ItemThe metaboloepigenetic regulation of preimplantation embryo development and fetal programming by ketone bodies( 2022)Diet-induced nutritional changes in the maternal reproductive tract elicit embryonic adaptations to maintain growth and survival, changes which nonetheless compromise long-term child and adult health via developmental programming. This is plausibly facilitated by metaboloepigenetic mechanisms, whereby metabolic sensing of nutrient availability coordinates epigenetic regulation of gene expression in a developmentally persistent manner, affecting short- and long-term embryo physiology and health. Despite little evidence for its efficacy and developmental safety, the very high fat, low carbohydrate ketogenic diet (KD) is increasing in popularity as a ‘fertility diet’ worldwide. In line with the hypothesis that metaboloepigenetics links diet with embryo health, KD consumption elevates systemic concentrations of the ketones beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc), which in addition to their role as oxidative fuels, are epigenetic modifiers and may consequently affect embryo development. Therefore, to investigate the safety of a periconceptional KD for offspring health, it is necessary to determine the impact of ketones on the development, physiology, and viability of the preimplantation embryo. Using a mouse model, the impact of in vitro ketone exposure on preimplantation embryo development, physiology and viability was assessed. Exposure to BOHB alone perturbed blastocyst development, decreased glucose metabolism, and increased histone acetylation, predominantly impacting the trophectoderm cell lineage. This was further associated with implantation failure and delayed female fetal development post-transfer, suggesting preimplantation BOHB exposure is detrimental to development in a female-specific manner via a trophectoderm-mediated mechanism. Similarly, in vitro exposure to AcAc alone or AcAc + BOHB in combination did not impair morphological development of the blastocyst, however impacted glucose metabolism and cumulatively increased histone acetylation. Preimplantation AcAc + BOHB exposure also increased miscarriage rates and delayed female fetal development post-transfer. Consistently, RNA-seq analysis identified persistent and sexually dimorphic effects of in vitro preimplantation BOHB and AcAc + BOHB exposure on fetal liver and placental gene expression. BOHB downregulated cholesterol synthesis pathways in female placenta, indicating compromised placental function, possibly contributing to the observed impairments in pregnancy establishment and maintenance. Oxidative metabolism was downregulated in ketone-treated female fetal liver, highlighting that ketones also affect the inner cell mass lineage and exert persistent changes in metabolic function. Notably, X-linked genes were overexpressed in ketone-treated female tissues, alluding to a mechanism underlying the sexually dimorphic effects of ketones, plausibly via dysregulation of X-inactivation. Finally, a maternal periconceptional KD was confirmed to elevate BOHB concentrations within mouse oviduct fluid, concomitant with delayed in vivo blastocyst development and altered trophectoderm-specific histone acetylation, corroborating the findings from prior in vitro studies. The data within this thesis provide a comprehensive analysis of the negative impact of ketone exposure on embryonic development and viability, confirming that metaboloepigenetic processes within the preimplantation embryo are sensitive to ketones, and provide mechanistic insight into sex-specific developmental programming. These findings suggest that periconceptional ketogenic diet consumption may be detrimental to long-term health, such that a KD during the periconception period is inadvisable. These findings may provide essential guidance for professional recommendations regarding dietary choices to improve fertility without increasing the time to pregnancy or compromising child health.
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ItemUsing indigenous microinvertebrates to assess the environmental impacts of soil pollution in Antarctica( 2022)Modern humans have had a continuing presence in Antarctica since at least the 1800s, and this presence has come with an environmental footprint. Since the introduction of the Madrid protocol in the 1990s, the Antarctic Treaty has ensured the protection of the environment from current and future impacts of human activity and enshrines a responsibility to remediate legacy contamination. Ice-free areas of Antarctica only account for a small fraction (<0.5%) of the landmass of the continent but are the location of >94% of current permanent stations. The concentration of human activity in these areas disproportionately exposes them to the impact of our activities. Ecotoxicologically-derived sensitivity estimates for endemic species can be used to assess the impacts of soil contamination on terrestrial Antarctic environments, informing risk assessment and land management for environmental protection. Prior to the work completed herein, there was little ecotoxicological information available for indigenous terrestrial microinvertebrates from Antarctica and none for tardigrades and rotifers. This thesis investigated the use of endemic terrestrial rotifers and tardigrades from Antarctica to assess the impacts of metals (Cu and Ni) present in the environment from historic waste disposal sites on terrestrial Antarctic ecosystems. The thesis was split into two stages: an initial stage in which cultures of terrestrial Antarctic microinvertebrates were established and life history traits recorded, and a second, in which the sensitivity of these microinvertebrates to Cu and Ni was determined in soil-based media. Soil based media were used to model the physiochemical conditions experience by the terrestrially-sourced rotifers and tardigrades in the environment. The bdelloid rotifer 'Habrotrocha' sp. and tardigrade 'Acutuncus' sp. were successfully isolated from moss and soil samples and cultured in two soil-based growth mediums, a soil elutriate, and a balanced salt solution (BSS). Both BSS and soil solution were suitable growth media for 'Habrotrocha' sp. and 'Acutuncus' sp., and moderate dilutions of soil elutriate were optimal for rotifer population growth. This was the first example of a soil-based medium being used to culture tardigrades or rotifers. The life history traits of the rotifer 'Habrotrocha' sp. were also assessed and found that they were long-lived compared to equivalent rotifers from the literature, with similar reproductive output across their lifespan. A meta-analysis conducted on the interaction between bdelloid rotifer life history traits and culturing temperature found that rotifer longevity and time to reproductive maturity were strongly negatively correlated with culturing temperature, expanding our knowledge of these organisms. The sensitivity of three terrestrial Antarctic microinvertebrate species to contaminants was assessed throughout this thesis, two bdelloid rotifers 'Philodina' sp., and 'Habrotrocha' sp, and one or more species of tardigrade, from the Family Hypsibiidae. 'Philodina' sp. directly isolated from environmental samples exposed to Cu in a water medium were highly sensitive compared to other bdelloid rotifers and demonstrated apparent toxicant-induced cryptobiosis as a novel endpoint for sensitivity estimates, a first for these taxa. In contrast, 'Habrotrocha' sp. and 'Hypsibiidae' sp. were exposed to Cu and Ni in a soil elutriate, demonstrating high tolerance to both metals compared to equivalent temperate rotifers, and similar microinvertebrates (e.g. nematodes) from the literature. The procedures and initial response data presented in this thesis will contribute to the establishment of environmental quality guidelines for soil contamination in Australia’s Antarctic Territory. The procedures can also be utilised for terrestrial microinvertebrates from other climates, assisting in incorporating more species into environmental risk assessments.
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ItemBuilding a wall: Developing small molecule biosensors to visualize cell wall biosynthesis and untangling mechanisms underlying nucleotide sugar transport( 2022)The cell wall is one of the main energy sinks in plants constituted of many polysaccharides and glycoproteins. The synthesis of most polysaccharides and the proteins glycosylation occur in the Golgi apparatus. The nucleotide sugars are the precursors of the cell wall building blocks. These molecules are biosynthesized in the cytosol from sugars of the primary metabolism and are transported from the cytosol to the Golgi by the nucleotide sugar transporters (NSTs). The glycosyltransferases (GTs) then consume these nucleotide sugars to produce polysaccharides and the glycoproteins. Although we have a generalized overview, our knowledge of the exact roles of NSTs and nucleotide sugars in regulating the cell wall synthesis is still sparse. The recent determination of the crystal structures of two NSTs, the GDP-D-mannose transporter VRG4 from yeast and the orthologue of CMP-sialic acid transporter from maize indicate that conformational changes occur during the transport process. Therefore, NSTs are excellent candidates to develop fluorescent proteins-based sensors to study the flow of nucleotide sugars. In chapter II, our aim was to generate fluorescent protein- biosensors based on the UDP-XYLOSE TRANSPORTER 1 (UXT1) to follow the import of UDP-D-xylose into the Golgi in vivo as a proxy for xylan and xyloglucan biosynthesis. We show that the UXT1-based FRET sensors maintain the physiological localization of UXT1 in planta, while transporting UDP-D-Xyl in vitro. We also designed ratiometric sensors based on the sfGOMatryoshka. However, these sensors were found to disrupt the Golgi localization of UXT1. In chapter III, we aimed to probe the existence of protein complexes involved in arabinosylation and study the regulation of the arabinosylation pathway. Investigating higher order mutants of the UDP-ARABINOFURANOSE TRANSPORTERs (UAfTs), allowed us to decipher the relative contribution of each of the UAfTs to arabinosylation. These newly generated mutants in combination with other mutants of the arabinosylation pathway led us to propose a mechanistic model to explain a glucose hypersensitivity phenotype in the dark. Phenotypic assessment of nucleotide sugar levels and hypocotyl elongation allowed us to make progress towards dissecting the role of cytosolic UDP-GLUCOSE EPIMERASE 1 and 3 in regulating nucleotide sugar metabolism. Finally, using affinity purification, split-ubiquitin assays and in silico co-expression approaches, we unravel putative NST-GT complexes and suggest that these complexes also involve nucleotide sugar interconverting enzymes.