Given the increasing frequency of climate change driven coral mass bleaching and mass mortality events, intervention strategies aimed at enhancing coral thermal tolerance (assisted evolution) are urgently needed in addition to strong action to reduce carbon emissions. Without such interventions, coral reefs will not survive. The seven chapters in my thesis explore the feasibility of using a host-sourced bacterial probiotic to mitigate bleaching starting with a history of reactive oxygen species (ROS) as a biological explanation for bleaching (Chapter 1). In part because of the difficulty to experimentally manipulate corals post-bleaching, I use Great Barrier Reef (GBR)-sourced Exaiptasia diaphana as a model organism for this system, which I describe in Chapter 2. The comparatively high levels of physiological and genetic variability among GBR anemone genotypes make these animals representatives of global E. diaphana diversity and thus excellent model organisms. The ‘oxidative stress theory for coral bleaching’ provides rationale for the development of a probiotic with a high free radical scavenging ability. In Chapter 3, I construct a probiotic comprised of E. diaphana-associated bacteria able to reduce oxidative stress by neutralizing free radicals such as ROS. I identified six strains with high free radical scavenging ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae, and Micrococcaceae. In parallel, I established a “negative” probiotic consisting of closely related strains with poor free radical scavenging capacities. The application of this probiotic to mitigate the negative impacts of exposure to a simulated heat wave was tested in Chapter 4. There was no evidence for improved thermal tolerance in E. diaphana. Changes in the relative abundance of anemone-sourced Labrenzia provided evidence for its integration in the E. diaphana microbiome. Uptake of other probiotic members was inconsistent and probiotic members did not persist in the anemone microbiome over time. Consequently, the failure of the probiotic inoculation to confer improved thermal tolerance may have been due to the absence of probiotic bacteria for the full duration of the experiment. Importantly, there were no apparent physiological impacts on the holobiont following inoculation, thus showing that shifting the abundance of native anemone microbiome members was not detrimental to holobiont health. Further, I found no evidence for an increase in ROS in the E. diaphana holobiont when it was exposed to heat. Some of the most compelling evidence in support of the ‘oxidative stress theory of coral bleaching’ comes from three published studies that expose corals, cultures of their algal endosymbiont, or E. diaphana to exogenous antioxidants during thermal stress. To confirm that ROS is the main driver behind thermal bleaching in E. diaphana, I replicated these previous experiments with novel methods that allowed a more accurate quantitation of ROS, and found that dosing with exogenous antioxidants (mannitol and ascorbate plus catalase) mitigates bleaching in E. diaphana, with no correlation between bleaching and increased ROS (Chapter 5). A serendipitous finding was that the E. diaphana bacterial community diversity can be rapidly reduced when anemones are reared in sterile seawater, making this model suitable for testing the efficacy of microbial restructuring strategies (Chapter 6). Taken together, the work from my PhD has shown that ROS scavenging varies among anemone-associated bacteria and that a high ROS-scavenging probiotic can be developed. Further, my findings have unveiled several main knowledge gaps that need to be filled before probiotics can be implemented, including administration strategies and choice of probiotic bacteria that maximise the maintenance of probiotic communities over time and a direct measurements of ROS in bleaching corals (Chapter 7).

Low or suboptimal temperature stress is one of the primary abiotic conditions limiting the growth and productivity of economic crops in many regions of the world. Wheat is one of the major crops in Australia, it is grown during winter to avoid hot summers and they flower in early spring. The sensitive flowering stage of wheat is therefore frequently exposed to spring frost. In Australia, the frequency of spring frosts during the flowering stage has increased significantly since 1960, and the reoccurrence of frost events led to an estimated $360 million of losses in the Australian wheat industry per annum. It is therefore important for breeders to minimize the loss via the development of more chilling/frost-tolerant wheat varieties, especially during their reproductive stages. Two approaches could be employed to achieve this goal. The first one is by employing metabolomics approaches to understand the underlying molecular mechanisms involved in cold responses of wheat upon cold stress. The second approach is via bioengineering of cold responsive genes into wheat to create chilling/frost-tolerant varieties. With this in mind, my PhD study was carried out with three main objectives. The first objective was to investigate and understand metabolic traits involved in the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The cold-sensitive variety used in this study was Wyalkatchem and the cold tolerant variety used was Young. The second objective of this study was to identify potential metabolite and lipid responsible for chilling tolerance in the two studied wheat varieties. The third objective was to evaluate the potential of REIL (Required for isotropic bud growth1 – like) protein as cold acclimation factor in Arabidopsis thaliana for potentially enhancing wheat cold tolerance. Chapter 1 consists of a review of the recent literature covering cold stress responses (physiologically and metabolically) of plants and how plants adopt to cold stress. It describes how metabolomics and lipidomics can be used as promising tools to decipher cold stress responses in wheat and discuss the role of cold-induced genes to increase cold tolerance in plants. The targeted protein in this study, REIL, as a new potential cold acclimation factor in Arabidopsis thaliana and wheat is also reviewed. To achieve the first and second objectives of this study, work described in Chapter 2 was conducted to investigate the cold acclimation of two Australian wheat varieties with contrasting cold tolerance using targeted metabolomics and lipidomics approaches. The selected cold-sensitive spring wheat variety used in this study was Wyalkatchem and the selected cold-tolerant spring wheat variety was Young. Samples of flag leaves and spikes at the young microspore stage were collected and analysed in this study. The results obtained provide us with a better understanding of the cold responses of wheat, and pointed out the potential of several sugars, amino acids, amines and glycerolipids to confer cold-tolerance to the Young variety. The outcomes gained from this study have been published in Cheong et al., (2019) for the study on flag leaves, and in Cheong et al., (2020) for the study on spikes. The outcomes also pointed out the profound potential of lipid species as biomarkers that can be explored to distinguish the two varieties. This further motivated us to expand the lipidomics study on the underground part of wheat, the roots (Chapter 3). There are limited cold stress studies on the lipidome of whole roots and to the best of our knowledge, no data are available on responses of specific root developmental zones. In Chapter 3, the lipid profiles of the spatial root zones derived from young seedlings of Wyalkatchem and Young grown at optimal, chilling and freezing temperatures were investigated. The outcomes indicate the involvement of not only glycerolipids in discriminating Young from Wyalkatchem, but sphingolipids are also involved in conferring cold-tolerance of Young. Next, to fulfil the third objective of this study, REIL, a protein that has been postulated to act as a potential cold acclimation factor in the mature leaves of Arabidopsis thaliana, was evaluated in roots in Chapter 4, followed by the evaluation of its potential in wheat in Chapter 5. REIL proteins have been postulated to be involved in late ribosomal biogenesis and affect the accumulation of 60S large subunits in the mature leaves of A. thaliana upon cold stress. To validate these roles in A. thaliana, a systematic analysis of roots grown at optimized and cold temperatures was conducted in Chapter 4. The outcomes substantiate the role of REIL proteins as a cold acclimation factor in Arabidopsis by being involved in ribosomal biogenesis during cold acclimation. In Chapter 5, three REIL homologs are found to be expressed in wheat. Evaluation of the REIL expressions in wheat subjected to cold stress through the re-analyses of published transcriptomics datasets show the potential cold and heat responsiveness of REILs. A real-time PCR analysis was then performed to evaluate the REIL expressions in Wyalkatchem and Young under cold stress, but no significant changes of expressions were observed in both varieties upon cold stress. It is then yet-to-be-known whether the cold acclimation function of REIL is conserved among dicots (A. thaliana) and monocots (wheat). Therefore, more in-depth investigation such as overexpression or silencing of the REIL expression in Australian spring wheat varieties is needed. The last chapter of this thesis (Chapter 6) summarizes the key results from each research chapter (Chapter 2 to 5) and also discusses the future directions and perspectives.

Tropical rainforests (TRFs), contain the most structurally complex and biodiverse plant communities of any terrestrial biome. Within these communities there are a variety of physical, temporal and physiological barriers that create unique challenges for effective long-distance seed dispersal. Endozoochorous seed dispersal by frugivorous animals is the most effective means of seed dispersal in these environments, particularly over long distances. The largest of these frugivores play a disproportionately important role as keystone seed dispersal vectors, processing large quantities of seed, transporting them over long distances, and swallowing seeds across the full spectrum of size allometries. For plant species with the largest of seed sizes, large-bodied frugivores constitute the only viable means of long-distance seed dispersal. In an era of unprecedented rates of TRF deforestation, fragmentation, and defaunation, the erosion of faunal diversity generally starts with the largest species. The extirpation of large-bodied frugivores from TRF creates a plethora of negative impacts for plants that ultimately impacts on their fitness and resilience. These impacts include shrinking mean seed dispersal distances, increases in the spatial clustering of young plant cohorts and a reduction in the genetic variability of plant populations as a whole. At the dawn of the 21st century, the vast majority of the world’s TRF exist in a fragmented and partially defaunated state. However, we have a very limited understanding of how defaunation, particularly for large-bodied animals, impacts the seed dispersal and population dynamics of tropical tree species. An ideal study system in which to generate broader insight for these knowledge shortfalls exists in Australia, in populations of the rare TRF tree, Ryparosa kurrangii B.L. Webber (Achariaceae). Restricted in range to just three lowland valleys of the Daintree region of Northern Queensland, R. kurrangii has many traits that make it amenable to addressing questions on plant-animal seed dispersal mutualisms. First, due to the large seed size and fleshy fruit of R. kurrangii, it was theorised that the species was adapted to seed dispersal by large-bodied frugivores, and therefore would be sensitive to changes in the population structures and foraging habits of frugivorous fauna. Second, the only functional long-distance seed disperser of R. kurrangii is the southern cassowary (Casuarius casuarius johnsonii). Cassowaries are the only extant frugivores with a gape-width large enough to swallow R. kurrangii seeds and pass them intact. Third, earlier work conducted in three fully-mapped study populations of R. kurrangii identified a gradient of recent anthropogenic disturbance between sites. These study populations have a history of regular measurement and provide an ideal basis for demographic studies of tree populations across their entire ontogeny. To better understand the links between anthropogenic disturbance, seed dispersal, resource availability and plant demography, research was conducted simultaneously on a number of facets of R. kurrangii life-history. To understand the basic population dynamics of the species, studies of demographic changes, recruitment rates, mortality, growth rates and maximum lifespan were conducted. Ryparosa kurrangii were found to grow and recruit exceptionally slowly, and could have lifespans of up to c. 600 years. High mortality within mature tree cohorts over the monitoring period make it seem likely that the number of reproductive trees will decline and will not be offset by growth of small individuals into taller height cohorts. Using growth models from repeated measures of R. kurrangii it was determined that almost all reproductively mature R. kurrangii are likely to have recruited prior to European settlement of the region (c. 85 years of age). To understand how the life-history processes of R. kurrangii populations were influenced by within-population and environmental conditions, the patterning of tree distributions and population attributes were analysed using spatial point processes. The spatial aspects of population recruitment, growth, reproduction and mortality, as well as their interactions, were assessed and compared between populations. Hot-spots of seedling recruitment were identified, but were not found in areas of high average sapling growth rate. It was therefore unlikely that superior site conditions were a strong spatial determinant for recruitment likelihood. Instead, recruitment hot-spots occurred in areas where high concentrations of seedlings had been previously recorded, and, that were also close to fruiting trees. This observation, coupled with the landscape scale clustering of mature stands of R. kurrangii trees, suggest that long-distance seed dispersal was uncommon in all populations, and had been so for many centuries. To understand how the visitation rates of vertebrate fauna, particularly those of cassowaries and introduced pigs (Sus scrofa; a known seedling antagonist) varied between populations, a camera trapping program using passive infra-red cameras was conducted over three years. Cassowary visitation was not consistent throughout the year, and varied in frequency between R. kurrangii populations, with visitation rate scaling with the ranking of anthropogenic disturbance. Moreover, cassowaries were not frequently recorded during peak R. kurrangii fruiting periods, indicating that prolific fruiting of R. kurrangii trees were not strong incentives for site visitation. Feral pigs were the most commonly detected animal species in the study, and were consistently detected throughout the year. Finally, to understand whether there was a ‘smoking gun’ signature of dispersal limitation in any R. kurrangii stand, population genetic analyses using microsatellite markers were conducted on c. 1300 plants across all ontogenetic stages. FST estimates suggested that 7% of genetic variation was partitioned between populations, indicating moderate between-population differentiation. The finding was in close agreement with an earlier study that used different genetic markers. FIS estimates were calculated to be between 0.39 and 0.47 across populations, revealing some of the most extreme inbreeding reported for outcrossing tropical trees. Strong spatial genetic structuring (SGS) was also detected in all R. kurrangii populations and in all stages of tree ontogeny. Significant SGS implied that closely spaced plants in R. kurrangii stands were close genetic relatives. The extreme population genetic indices reported for this species strongly indicate that effective long-distance seed dispersal has been severely limited in R. kurrangii populations for thousands of years. Evidence from the spatial clustering of trees at all stages of ontogeny, the lack of linkage between cassowary visitation and R. kurrangii fruiting season, and, the extreme inbreeding and spatial genetic structure in all populations points to a similar story. Instead of measuring a recent disruption to frugivore visitation from the disturbance caused by European settlement of the Daintree, the magnitude of observed patterns are more readily explained by the loss of one or more large-bodied seed dispersal agents in the distant past. Despite the apparent capability of extant cassowaries to be efficient and high-quality dispersal agents to R. kurrangii, it appears that currently they are not effective in consuming and dispersing their seed. These findings have significant implications not only for the conservation of R. kurrangii, but also for the broader conservation of rainforest trees in systems where it is assumed generalist frugivores are providing adequate seed dispersal services. Ryparosa kurrangii could join a growing list of plant species with anachronistic adaptations for dispersal by extinct frugivores. It is postulated that the original co-evolved dispersal agent(s) may have been driven to extinction through a much older anthropogenic disturbance, that of the arrival of the first Aboriginal peoples to North Queensland more than 40,000 years ago. The implication that extant large-bodied frugivores that are apparently capable of dispersing large-seeded TRF plants, but that are not adequately fulfilling a keystone dispersal role, is a cause for concern. Many plant species across the world that are presumed to be adequately serviced by local fauna may similarly be under-dispersed to the point of inbreeding depression and population collapse. If the species diversity and community complexity of tropical rainforests are to be maintained beyond the next few generations of trees, a shift in conservation tactics is likely needed. Future conservation management will need to more strongly focus on identifying and retaining key plant-animal interactions, as well as mitigating for those that are now long gone.

This thesis is composed of three data chapters and a general introduction and discussion. Each chapter, except for the general introduction and the general discussion is composed of an introduction, materials and methods, results, discussion, and conclusions. The aim of this study was to assess the vulnerability risk to fishing and climate change stressors of 106 species of chondrichthyans with ≥10% of their distribution within the EEZ off western Mexico. For my analysis, I determine the vulnerability of the chondrichthyan species inside the Gulf (GCI) and compare these results with those for two other contiguous broad regions with different oceanographic conditions, the region around the entrance to the Gulf of California (GCE) and Mexico’s remaining Pacific waters (MPW). I have built on existing approaches to provide, in a single framework, a vulnerability analysis and risk assessment of the Mexican chondrichthyan (sharks, rays, skates and chimaeras) fauna by combining three components of vulnerability risk to climate change (exposure, sensitivity and adaptive capacity) (ESA), together with three components of vulnerability risk to fishing stressors (exposure, productivity, and susceptibility) (EPS). Here, vulnerability is expressed as the risk of marked reduction of the population of chondrichthyans based on the knowledge of its biology and its exposure to stressors associated with fishing and climate change. For fishing stressors, I use the productivity of the chondrichthyan species, which is related to the maximum age of the species, and susceptibility, which derives from four parameters; availability, encounterability, selectivity, and post-encounter mortality. For climate change stressors, I use sensitivity, which has two parameters; rarity and habitat specificity as species attributes that contribute to this, and adaptive capacity. Adaptive capacity involves distributional flexibility and trophic level as relevant attributes. I assigned each species to one of six ecological groups (EGs), which is a flexible and novel way to allocate a large number of species based on habitat use, depth strata (shelf-inshore and shelf-offshore), habitat dependence (freshwater, reef substrate, and sandy substrate), and lifestyle (demersal or pelagic). For fishing stressors, I analyzed data sets from 2006 to 2017 for the prawn trawl fishery, the elasmobranch fishery (artisanal and semi-industrial) and for the sardine fishery, and published information on the sport-recreational fishing. These fisheries have the potential to reduce the size of the population of a chondrichthyan species by altering the mortality rate in the regions where the fisheries operate. I then characterised the fishing stressors in terms of fishing methods and the bathymetric range of deployment of the fishing gear (Chapters 1, 3 and 4). For climate change stressors, I obtained data sets from several sources to show trends in the past oceanographic conditions and how they may vary in response to climate change. I then characterised the oceanography of the Gulf of California and adjacent waters. My assessment is based on observed changes from 1960 to 2017, and projected changes. In the period from 1960 to 2017 two important phenomena that warm the sea surface water occurred; “El Nino” and “El Blob”. The latter is a phenomenon related to a warm mass of water as a result of high levels of atmospheric pressure and of which origin is detected in the Gulf of Alaska in 2013. The name “Blob” echos the 1958 horror film which describes a character that keeps growing as it consumes everything in its path just as this warming event did (Cornwall, 2019). The “El Blob” was detected until several months later 2013, so it is unknown whether “El Blob” can occur again with the same or higher intensity. The other timescales are based on projected changes by 2055 and by 2099 using low (2.6), medium (4.5) and high (8.5) emissions scenarios from the RCP (representative concentration pathway) family. Because of a temperature gradient in coastal waters increasing from north-western Mexico to southwestern Mexico (Chapter 2), I established ten contiguous ‘subregions’ in these waters (Chapter 4). This allowed me to evaluate the risk associated with the attribute 'distributional flexibility' of the chondrichthyan species and to determine thermal tolerance range categories as follows: all waters (AW), cool waters (CW), warm waters (WW) or Gulf of California water (GoCw). These categories provided a basis for projecting how chondrichthyan distributions might change in response to climate change. I identified a total of 54 species of sharks, 48 species of rays and 4 species of chimaeras, which belong to 3 superorders, 12 orders, and 33 families. Based on the thermal tolerance range indicated by the current presence-absence of each species in the subregions, a total of 35 chondrichthyan species are distributed in all Mexican waters (AW), suggesting the species are adapted to the full range of temperatures currently occurring in Mexican waters. The majority of these are commercial shark species and these are the least likely species to redistribute out of Mexican waters as waters warm progressively northward as climate change progresses. A total of 31 species were classed CW (i.e., favouring cooler waters) and likely to reduce their distributional range northwards as Mexican waters warm in response to climate change. The majority of these are also commercial shark species. On the other hand, 34 species of chondrichthyans were classed WW (i.e., favouring warm waters), and are likely to expand their distribution northwards within Mexican waters. The majority of these are ray species, some of them of commercial importance. One species of shark, one species of ray and one species of chimaera are distributed only in the GoC waters, and another species of shark and two species of rays are distributed in only inside and outside the GoC in the adjacent MP-C subregion. The ecological groups (developed for all three regions) are shelf-inshore, shelf-reef, shelf-sand (<75 m), shelf-sand (75–150 m), pelagic waters and bathyal (>150 m). A total of 46 species were allocated to the ecological group ‘shelf-sand (<75m)’, 14 species were allocated to the ecological group ‘shelf-sand (75–150 m)’, and 22 species to the ecological group ‘pelagic waters’. Some of these species are demersal and others swim near the bottom or may swim up in the water column. A total of 19 species of chondrichthyans are in the ecological group ‘bathyal (>150 m)’, one species is in the ecological group ‘shelfinshore’, and 4 species were allocated to the ecological group ‘shelf-reef’. Vulnerability risk varies among the current chondrichthyan species, among ecological groups and among fishing and climate change stressors. For total vulnerability to fishing stressors, there were 10 species in the GCI and GCE regions, and 40 species in the MPW region at medium vulnerability risk. I determined 33 species in the GCI and GCE regions, and one species in the MPW region were at high vulnerability risk. For climate change (CC) stressors in the whole of western Mexico, a total of 15 and 10 species were at medium vulnerability risk under the medium and high emissions scenarios, respectively, and 10 species were at high vulnerability risk under the high emission scenario. The species allocated in the EG shelf-sand (<75 m) are highly vulnerable to the combination of fishing and CC stressors in all three regions for all the CC scenarios. In contrast, the species allocated in the EG bathyal (>150 m) are at low vulnerability but varies for species allocated to the other EGs.

The world is rapidly urbanizing, transforming Earth’s natural habitats. While the loss of these habitats is the most visible impact of urban expansion, human population growth has also brought associated phenomena that have the capacity to negatively impact non-human animals. Two key anthropogenic phenomena associated with urban environments have become almost inescapable: anthropogenic noise and artificial light at night. Anthropogenic noise can now be heard in some of the most remote protected areas in the world, while, in the US alone, half of all land mass is exposed to unnatural levels of light at night. There is growing evidence that these environmental ‘pollutants’ have negative impacts on the behaviours and fitness of urban wildlife. In this thesis, I focus on the potential of these anthropogenic pollutants to disrupt cognitive processes and sleep in an urban-adapted species, the Australian magpie (Cracticus tibicen). Wild magpies occupying territories across suburban Melbourne that were exposed to varying levels of urban noise performed similarly on cognitive tasks. Variation in cognitive performance was best explained by age rather than the amplitude of noise to which birds were exposed on a daily basis. I found some evidence that higher sound levels may impair cognitive development, but the effect was observed in only one of four cognitive tasks presented. In captivity, the performance of magpies on similar cognitive tasks was compared under two conditions (experimental exposure to realistic traffic noise, compared to a quiet control). I again found no difference in cognitive performance in the presence or absence of noise. They did, however, perform better on tasks the second time they experienced them, regardless of noise. Finally, I explored the effect of urban noise and two different colours (blue-rich and blue-reduced) of artificial light at night on sleep in magpies. Magpies exposed to a 24-hour recording of urban noise spent more time awake and less time in non-rapid eye movement (non-REM) and REM sleep throughout the night. Likewise, magpies exposed to artificial light at night spent less time asleep, and their sleep was more fragmented. In addition, blue-rich lighting had a greater effect on sleep than did blue-reduced lighting. My findings shed light on some of the putative pathways by which anthropogenic noise and artificial light can affect key biological processes in suburban wildlife, but also illustrate that not all processes are necessarily detrimentally affected.

Fungal species impact humans in many areas, some positively and others negatively. Some fungi are agents of disease in plants or animals, yet others find diverse beneficial uses in industry and food production. A greater understanding of fungal biology is a pressing requirement because it will allow us to ameliorate the negative effects of certain species and enhance currently beneficial interactions, as well as to identify new uses for fungi. With this in mind, this thesis has focused on the development of new molecular tools and approaches that will improve capabilities for gene characterisation and phylogenetic inference in fungi. This work has extended across several species including the canola pathogen Leptosphaeria maculans, the heat tolerant ascomycete Paecilomyces variotii and the species in the order Mucorales. In L. maculans I utilised novel tools including CRISPR-Cas9 to identify pathogenicity genes. After evaluating the efficiency of identifying pathogenicity genes using a combination of RNA-sequencing based prediction and CRISPR-Cas9 targeted disruption of putative pathogenicity genes, I turned to a more conventional approach using T-DNA mutagenesis and forward genetics to screen for mutants with altered pathogenicity. I paired this with whole genome sequencing to identify T-DNA integration events, and in this way identified three new pathogenicity genes, one encoding a Sit4 Associated Protein (SAP), one encoding a flavoprotein, and one encoding a heat repeat protein. In Paecilomyces variotii I developed all the tools required to work effectively at the genetic level. These include two genome sequences and techniques for transformation, targeted gene disruption and sexual crossing. Using these tools, I then examined interesting aspects of the biology of this organism including discovering Repeat Induced Point (RIP) mutation for the first time in the Eurotiales and uncovering the genetic basis for the biosynthesis of the secondary metabolite viriditoxin. In the final chapters of this thesis I describe some of my work uncovering diversity among Australian Mucorales species. This has included the discovery of a new species of Pilaira, a new species of Syncephalastrum and a comprehensive examination of the genus Backusella.

Colour polymorphism, the co-existence of multiple heritable colour morphs within an interbreeding population, is thought to promote rapid phenotypic evolution and speciation. This is based on the importance of colour signals in reproductive isolation in combination with the underlying genetic architecture of polymorphism, where morphs are predicted to be governed by few genes of major effect. This prediction is supported by empirical data and stems from how colour morphs often differ in suites of co-adapted traits. During secondary contact between populations that differ in morphs, there is expected to be a high probability of genetic incompatibilities between morphs due to a breakdown of adaptive genetic correlations. Furthermore, colour signal divergence may also be accompanied by changes in behaviour and/or mating preferences leading to incompatibilities between populations which differ in morphs. These factors together may facilitate the formation of reproductive isolation and ultimately lead to speciation. In this thesis, I investigated divergence and the outcome of secondary contact between lineages of the tawny dragon, Ctenophorus decresii, which differ in morph number and type. Ctenophorus decresii is a sexually dimorphic agamid lizard endemic to South Australia, and comprises two genetically distinct and geographically structured lineages: northern and southern. I tested for differences in colour vision between the lineages, which differ in a sexual signal, male throat coloration, particularly in the absence or presence of ultraviolet (UV) reflectance. The northern lineage is colour polymorphic with four discrete throat morphs which lack significant UV reflectance: orange, yellow, orange-yellow (orange centre surrounded by yellow), and grey. Southern lineage males are monomorphic with blue throats and a strong UV reflectance peak. Male throat coloration is an important intraspecific sexual signal, as it is emphasised in territorial and courtship displays. I investigated whether lineages differ in visual sensitivity to UV wavelengths by measuring retinal opsin protein expression of four cone opsin genes (SWS1, SWS2, RH2, LWS) using droplet digital PCR. I found that lineages did not differ in gene expression of the four opsins and discussed this in the context of conserved visual sensitives in terrestrial systems. The lineages meet in a contact zone where multi-locus genetic data suggested the presence of hybrids and potential barriers to gene flow. Using extensive field surveys, male phenotype data, genomic single nucleotide polymorphisms (SNPs), and a mitochondrial (mtDNA) marker, I investigated the outcome of secondary contact between the lineages. Furthermore, I captive-bred pure and first generation (F1) hybrid offspring to characterise colour traits independent of exogenous selection. I found that the contact zone is narrow and several generations old with no parental forms or F1 hybrids present. The northern mtDNA haplotype was prevalent in hybrids, and there were high frequencies of backcrossing to the northern lineage but not to the southern lineage, indicating genetic incompatibilities. The northern throat polymorphism was maintained, without any loss of morphs, whereas the southern throat morph was absent. This contrasted with the more intermediate throat phenotype of captive-bred F1 hybrids, particularly in ultraviolet reflectance, suggesting strong selection for the northern throat phenotype within the contact zone. The viability and fitness of F1 hybrids have consequences for contact zone dynamics, and ultimately whether species boundaries are eroded or maintained. I performed pure and reciprocal cross F1 hybrids in a laboratory setting and measured parental reproductive traits and offspring fitness traits. I found that northern females have a higher reproductive output with more, larger clutches per breeding season and lower embryonic mortality. Although pure and hybrid offspring did not differ in individual fitness traits, hybrids produced from a combination of northern females and southern males exhibited higher fitness in more categories (i.e. growth rate, bite force, sprint speed). These factors in combination may contribute to the prevalence of northern lineage mtDNA haplotypes in the contact zone. Finally, I taxonomically separated the northern and southern lineages of C. decresii sensu lato on the basis of differentiation in morphology and male coloration, genetic divergence with restricted gene flow, and geographic structuring. This revision results in C. decresii sensu stricto (previously southern lineage) and C. modestus (previously northern lineage). I evaluated morphological traits of the type specimen of Amphibolurus modestus (Ahl 1926), previously a synonym of C. decresii sensu lato, and determined that it represented a specimen of the northern lineage. Therefore, I formally re-instated and re-described Ctenophorus modestus (Ahl 1926). The addition of this species to the C. decresii species group, which now comprises six species, supports the notion that geographic divergence in male coloration is an important component to speciation in this group.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate neurotransmission at cholinergic synapses. The nAChRs are mainly expressed in the central nervous system and are highly conserved across a wide range of insect species. Neonicotinoids and spinosyns are two classes of insecticide that target nAChR subunits to kill pest insects. Mutations in genes encoding several nAChR subunits in various insect species, as well as in Drosophila melanogaster, have been documented as conferring insecticide resistance. Chemical control including insecticides has been a key tool in controlling pest insects. The cycle of insecticide use, resistance evolution and insecticide replacement has been continuing for the past decade, leading to many pest species carrying resistance to multiple classes of insecticides. This thesis examines the interplay between different insecticides and nAChR mutations that are associated with resistance to one insecticide but result in hypersensitivity to another, a phenomenon called negative cross-resistance. The negative cross-resistance relationship presents insecticides that could complement current rotation strategies for resistance management, and this warrants further analysis to understand the mechanism. Examination of loss-of-function mutations on the nAChR subunits in this thesis, confirmed the previous identification of the Dalpha1 and Dbeta2 subunits as targets for neonicotinoids, as well as the Dalpha6 subunit as a target for spinosyns. This study also identifies the Dalpha2 subunit as an additional target for imidacloprid. Importantly, mutations on these subunits were also associated with insecticide hypersensitivity, suggesting negative cross-resistance. The neonicotinoid-resistant, Dalpha1 mutants were hypersensitive to spinosyn, except for a full knockout allele, while the spinosyn-resistant, Dalpha6 mutants were all hypersensitive to neonicotinoids. Additionally, negative cross-resistance was found between two neonicotinoids, nitenpyram and imidacloprid in the Dalpha2 mutants. Analysis of different allelic variations at the gene encoding these subunits indicates that this is not an allele specific phenotype. Combining the negative cross-resistance relationship and analyses of molecular changes induced in the nAChR subunits mutants, our study initiated to characterise the changes at the synapse that underlie the negative cross-resistance phenotype. A mechanism involving nAChR compensatory changes in levels of another receptor subunit/subtype was hypothesised to cause the phenotype. Following measurement of transcriptional changes and subunit protein changes, the study classified few correlations between nAChR subunit expressions and the negative cross-resistance, and these vary between the mutants suggesting other possible route(s) for the insecticide hypersensitivity. A genome-wide differential gene expression analysis in specific neuronal cell types of larval brain revealed differentially expressed genes in the Dalpha1 and Dalpha6 mutants. Interestingly, gene ontology enrichment analysis indicates dysregulation of cellular processes, including oxidative stress, protein trafficking and proteasomal degradation pathways in the mutants, that may contribute to the insecticide hypersensitivity. Dysregulation of oxidative stress may predispose the nAChR mutants to further insecticide-induced increase in oxidative levels. Finally, blocking dynamin-mediated endocytosis and proteasome activity, using chemical inhibitors, showed protection against larval movement reduction following imidacloprid and/or spinosad exposure. These findings indicate that the relatively straightforward phenotypic observation of insecticide hypersensitivity in response to loss of a receptor subunit is most likely underpinned by several complex changes in neurons, altering the sensitivity of their response to insecticides and their capacity to cope with downstream effects of insecticide exposure.

Soil salinity is an important problem that impacts agriculture globally. A sustainable approach for improving productivity is to adopt beneficial microorganisms to enhance the supply of soil nutrients to plants in stressful environments. Our work is showing that the fungus Trichoderma harzianum T-22 enhances barley growth and nutrient uptake in saline conditions. The fungus symbiotically lives inside the roots and triggers beneficial biochemical and metabolic changes. This project has broad implications for applying beneficial plant-microbe interactions to improve agricultural productivity. Chapter 1 covers the history and performance of endophytic fungi applied to crops as an alternative or supplement to the use of plant genetics or soil management to alleviate salt stress in crops. We focus primarily on root-associated microorganisms. The root-soil zone is the first point of defence for the plant against salt moving into the plant with the transpiration stream. It has dynamic biogeochemical processes driven by diverse metabolites released by the plant root and associated soil microorganisms. Fungal endophytes associated with some crops not only protect against plant pathogens and pests but also impart strong tolerance against several abiotic stresses in crops, including salinity. This is achieved via inducing systemic resistance, increasing the levels of metabolites such as pathogen protectants and osmolytes, activating antioxidant systems to prevent damage caused by ROS, and modulating plant growth phytohormone levels. Colonization by endophytic fungi improves nutrient uptake and maintains ionic homeostasis by modulating ion accumulation, thereby restricting the transport of Na+ to leaves and ensuring a low cytosolic Na+:K+ ratio in plants. This literature review has been submitted after external review to the journal Plant and Soil for publication. Following there is an addendum (Section 1.6) covering investigations and application of the endophyte-plant interactions using in metabolomics and lipidomics. The addendum aims to provide an overview of the necessity to investigate plant-fungal interactions and its influence on plant metabolism. Lastly, the chapter gives a brief description of the main experimental, technical and instrumental methods employed in this project. Chapter 2 determines the effect of the salt tolerant beneficial endophyte, Trichoderma harzianum strain T-22, on the growth and development of barley under optimal and saline conditions. Two barley genotypes were used in the study, cv. Vlamingh as a salt tolerant cultivar and cv. Gairdner as a salt sensitive cultivar. Barley was chosen as it is not only an agriculturally and industrially important crop but is also the most salt tolerant cereal crop. Two experimental setups were used for this study. In the first experiment, agar was used as the growth medium for plants. This was performed to observe and determine the effect of fungus on the growth of plants under controlled conditions. In the subsequent experiment, sandy loam soil was used as the matrix to grow plants and to emulate agricultural scenarios. Light microscopy was used to confirm the association of the fungus within roots. The results confirm the positive effect of this fungus under controlled and saline conditions in both experiments. Various parameters were measured to confirm the effects of salt and fungus on both genotypes under controlled and saline conditions. This study suggests that inoculation of salt sensitive plants with T. harzianum T-22 may ameliorate the effects of salinity and improve plant growth. Chapter 3 describes the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) by mapping metabolites and lipids using GC-MS for polar metabolites and LC-MS for lipids. This was performed to provide insights into the biochemical changes in barley roots treated with fungus during the early stages of interaction. T. harzianum increased the root length of both genotypes under controlled and saline conditions. The fungus reduced sugars in both genotypes and caused no changes in organic acids under saline conditions. Amino acids decreased only in cv. Gairdner in fungal-inoculated roots under saline conditions. Triacylglycerols (TAGs) were the substantially increased lipids in inoculated roots of both genotypes under saline conditions. This study shows that the fungus imparts adaptation or tolerance mechanism to cv. Vlamingh and in cv. Gairdner by remodelling lipids mainly from glycerolipids after salt stress. Chapter 4 examined the role of Trichoderma strain T-22 on two barley genotypes (cv. Vlamingh and cv. Gairdner) grown in saline soil as soil is an important substrate for Trichoderma. Biomass results described in Chapter 2 of this thesis clearly show the positive effect of this fungus on both genotypes under control and saline conditions as measured using several physiological parameters. Here, the aim of this part of study was to determine the metabolites and lipids modified in roots grown in saline soil following endophyte inoculation that are involved in conferring positive effects on barley plants as mentioned in Chapter 2. We employed gas chromatography and liquid chromatography both coupled to mass spectrometry to analyse metabolites and lipids in inoculated and uninoculated roots of both genotypes under control and saline conditions. Chapter 5 explains the application of mass spectrometry imaging (MSI), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence (XRF) to determine the spatial distribution of metabolites, lipids and a range of elements, such as K+ and Na+, in seeds of two barley genotypes with contrasting germination phenology (Australian barley varieties Mundah and Keel). This chapter was published in Front Plant Sci (Gupta et al. 2019). Following this, an addendum (Section 5.10) is added to this chapter where the effect of T. harzianum was measured on germination of four barley genotypes used in this project. The results showed that the fungus improves germination efficiency of the sensitive genotypes, suggesting its role in reducing the adverse effects of salt stress on germination and growth of the plant. The final Chapter 6 describes the application and importance of mapping the biochemical changes involving metabolites and lipids, imparted due to the inoculation of the fungus in roots of barley seedlings and provides a view on future perspectives of research of plant-fungal interactions for plant growth promotion which can useful for sustaining agricultural productivity.

The plight of insect populations around the world has gained increasing attention. A recent meta-analysis published in Science reported an average decline of terrestrial insect abundance by average 9% per decade since 1925 (van Klink et al. 2020). While this is a lower rate of decline than reported in earlier meta-analyses (Sanchez-Bayo and Wyckhuys 2019) it still suggests that many terrestrial insect species are under threat. The extinction of terrestrial insect species would severely affect agriculture and ecosystems due to the vital role that many species play in pollination, the recycling of organic matter, pest control and other ecosystem services. Insecticide exposure has been proposed to be one of the significant contributing factors for population declines of non-pest species. Insecticide contamination in biomes, resulting from intensive usage on agricultural crops, is likely to lead to exposures for many non-pest insect species. Low doses of insecticides are known to impact the fitness and behaviour of various insect species, but the underlying molecular, cellular, and physiological impacts of such doses in insects are not well defined. The absence of a mechanism that explains how low doses affect insects is an obstacle to ascertaining the extent to which insecticides may contribute to the demise of populations. The aim of this study was to scrutinize the impacts of low insecticide doses on the metabolism and physiology of the model organism Drosophila melanogaster in order to propose a mechanism to explain the impact of such doses on insect biology. Two insecticides were investigated in detail. The first of these is the synthetic neonicotinoid imidacloprid. Having been banned in the EU due to some evidence of a role in collapse in honeybee colonies, imidacloprid remains one of the most widely used insecticides in the world. The second insecticide is spinosad. Composed of two structurally similar natural fermentation products from the soil bacterium Saccharopolyspora spinosa, this insecticide is classified as organic and considered to be less harmful to beneficial insects. Both insecticides target evolutionarily conserved nicotinic acetylcholine receptors (nAChRs) in the Central Nervous System (CNS) of insects. nAChRs are pentameric ligand gated ion channels. Activation by the natural ligand, acetylcholine, leads to a flux of calcium, potassium or sodium ions into neurons, regulating a myriad of responses in the insect brain. The Drosophila genome encodes 10 nAChRs subunits (Dalpha1 to Dalpha7 and Dbeta1 to Dbeta3), meaning that there is a vast number of subunit combinations that could assemble to form functionally distinct receptor subtypes. Imidacloprid targets the Dalpha1, Dalpha2, Dbeta1 and Dbeta2, subunits, whilst spinosad targets the Dalpha6 subunit. Acute exposure to imidacloprid, at doses that do not kill Drosophila larvae, rapidly increased in the levels of reactive oxygen species (ROS) in the brain, most likely due to the sustained calcium flux into neurons caused by the interaction between the insecticide and its nAChR targets. This led to oxidative stress marked by mitochondrial dysfunction that in turn led to a significant decrease in energy (ATP) levels. While this process was initiated in the brain, lipid storage in the metabolic tissues (fat body, Malpighian tubules, and midgut) was affected. Transcriptomic analysis of the larval brain and fat body revealed a significant perturbation in the expression of genes involved in metabolism, oxidative stress, and immune response. Using genetic manipulations to elevate ROS levels exclusively in the brain, lipid storage was shown to be perturbed in the metabolic tissues, indicating that a ROS signal initiated in the brain radiates to other tissues. Severe damage to glial cells and neurons (i.e. neurodegeneration) was observed in the visual system of adults subjected to chronic low-dose exposure to imidacloprid. This precipitated a progressive loss of vision. Spinosad showed a different mode of action, blocking nAChRs and preventing calcium influx. The blocked receptors were shown to be recycled from the neuronal membranes through endocytosis. This mechanism led to an increase in the number and size of lysosomes in the CNS, characteristic of lysosomal storage diseases, which precipitates elevated generation of ROS by impairing mitochondrial activity and neurodegeneration. The high levels of ROS measured in the CNS after spinosad exposure, were associated with a cascade of phenotypes in metabolic tissues similar to the ones observed after imidacloprid exposure. Experiments examining the lipid environment in Dalpha6 knockout mutants (resistant to spinosad) indicated that impacts observed in the metabolic tissues of spinosad-exposed larvae are due to the interaction between Dalpha6 and spinosad. These data corroborate the hypothesis that impairments observed in metabolic tissues are triggered by a chemical signal from the brain, suggested to be a peroxidized lipid. Although there were some differences in the responses observed for the two insecticides (e.g. in transcriptomes and lipidomes), a similar cascade of processes was observed to be initiated following the elevation of ROS levels in the brain. A potent antioxidant, N-Acetylcysteine amide, strongly suppressed a range of phenotypes observed in both larvae and adults, indicating a causal role for ROS and oxidative stress. As the nAChR targets of these insecticides are conserved among insects, it is likely that similar impacts would be precipitated by exposures in other non-pest species, albeit at different doses. As insecticides from a wide range of chemical classes create markers of oxidative damage, the low dose mechanism of action observed for imidacloprid and spinosad may apply more broadly. This requires investigation. Considered together, the low dose impacts of imidacloprid and spinosad severely impair insect biology, without necessarily killing. These impairments could render insect species more vulnerable to the other major threats proposed to contribute to the decline of populations: climate change, habitat loss, pathogens, and parasites.

Adaptive radiation plays a significant role in the generation of biological diversity, and the advent of modern sequencing approaches has unlocked a new genomic perspective on this process. Genomic-scale data from the across the diversity of adaptive radiations can provide unprecedented resolution of the phylogenetic, biogeographic and molecular context of diversification. Murine rodents (Murinae: Rodentia) are a recent and rapid adaptive radiation that make up > 10% of mammal species. Murines have repeatedly colonised new geographic areas and island systems in the Eastern Hemisphere, frequently as a result of overwater transitions. Recurring adaptive radiation, ecological character displacement, and convergent evolution across Murinae make them an ideal model for studying adaptive radiation, especially in the Indo-Australian region. Within broader Murinae, the Hydromyini are a speciose Australo-Papuan radiation that diversified following an overwater colonisation from Sunda to Sahul ca. 8 Ma. Previous multilocus studies did not provide sufficient phylogenetic resolution of the rapid diversification of Hydromyini, and did not adequately sample taxa to reconstruct their complex biogeographic history. In addition to unresolved biogeography, the endemic Australian clade within Hydromyini has suffered the highest rate of recent mammalian extinction in the world. The rapid decline of Australian rodents is thought to be primarily the result of predation by feral cats, combined with other factors such as anthropogenic land clearing. There is little information about the pace of decline in eight species that went extinct on the Australian mainland in the last 150 years, and it is unclear whether these species had suffered longer term declines that predate the arrival of Europeans into Australia in 1788. To resolve these outstanding issues, I develop a novel exon capture approach for murine rodents. Firstly, I investigate the degree of congruent and conflicting phylogenomic signal in a rapid radiation, using genus-level relationships in the Hydromyini as a model example. My results show that in a number of cases, strong conflict is not reflected in branch support metrics obtained using either maximum likelihood or summary coalescent approaches. This result is significant, as it suggests that approaches commonly used to estimate support in phylogenomic data can fail to detect uncertainty in the face of underlying genealogical heterogeneity. Further leveraging this novel exon capture design, I generate a robust phylogenomic tree based on > 350 samples across the Australo-Papuan continent, including extant and recently extinct species in Hydromyini. With these data, I reconstruct the species-level evolutionary and biogeographic history of the Hydromyini across Sahul, recovering numerous examples of overwater colonisation between regions. Consistent with the geomorphological hypothesis that the New Guinea lowlands emerged after the orogeny of the Central Cordillera, I find evidence for increasing ecological opportunity in the Hydromyini from approximately 5 Ma. This first species-level phylogenomic study spanning the entire Sahul region provides a baseline example for future comparative studies that seek to reconstruct the biogeographic drivers of diversification in Sahul at a continental scale. Using exon capture and whole-exome sequencing data from extinct and extant species, I place recently extinct Australian rodents in a phylogenomic context for the first time. I recover no marked evidence of genetic erosion in five extinct species at the time of specimen collection, in comparison to extant species with present-day low allelic diversity. This indicates that the decline of recently extinct Australian rodents occurred extremely rapidly, and its onset likely did not predate European settlement. Additionally, my results taxonomically resurrect a species from extinction, Gould’s mouse (Pseudomys gouldii), which survived as a single island population in Shark Bay, Western Australia (currently classified as P. fieldi). Finally, I generate whole exome data from 38 species in the global radiation of Murinae to examine patterns of positive selection and convergent evolution. I uncovered pervasive positive selection across genes associated with diet, digestion and taste across Murinae, and increased rates of adaptive evolution in carnivores compared to omnivores. Limited evidence for molecular convergence in worm-eating specialists Paucidentomys and Rhynchomys suggests a role for developmental phenotypic control in this striking example of ecological convergence. Broadly, my results indicate that the pronounced ecological and phenotypic shifts that are hallmarks of adaptive radiations may also drive corresponding shifts in the pace and pattern of molecular evolution across the genome. Together, the work in this thesis is fundamental to the understanding of diversification, adaptation and extinction in the Australo-Papuan region, and provides an extensive genomic resource for future studies.

Ascorbate (ascorbic acid, vitamin C) is essential for both plants and mammals. Ascorbate is a reducing agent capable of donating electrons, enabling it to perform a range of biochemical functions, such as scavenging reactive oxygen species, assisting enzymatic activity, and reducing higher oxidative states of iron (Fe). In plants, ascorbate is the most abundant water-soluble antioxidant and plays a key role in many fundamental processes, such as photosynthesis, stress tolerance, and the transport of Fe. In humans, ascorbate is an essential micronutrient that must be obtained through diet and takes part in a range of important physiological functions, such as collagen synthesis, epigenetic programming, and Fe uptake in human digestion. Several pathways towards ascorbate biosynthesis have been proposed in plants, but there is only definitive evidence for the L-galactose pathway. The GDP-L-galactose phosphorylase (GGP or vtc2/5) gene encodes the first-committed and rate-limiting enzymatic step of the L-galactose pathway and represents the most promising candidate for increasing ascorbate biosynthesis in plants. In addition to transcriptional regulation, the translation of GGP is regulated through a highly conserved, cis-acting upstream open reading frame (uORF) in the 5’ leader sequence of the GGP mRNA. Developing strategies to increase ascorbate biosynthesis in rice (Oryza sativa L.) and wheat (Triticum aestivum L.), two of the world’s most important staple crops, has the potential to improve both food security and crop productivity. As part of this PhD project, two distinct metabolic engineering strategies were employed to increase ascorbate concentrations in rice: (i) constitutive overexpression of the OsGGP coding sequence (35S-OsGGP plants), and (ii) CRISPR/Cas9-targeted mutagenesis of the OsGGP uORF (uorfOsGGP mutants). Ascorbate levels were negligible in both 35S-OsGGP and uorfOsGGP brown rice, likely due to the decline of ascorbate levels in maturing grain reported in cereals; highlighting the challenge of increasing ascorbate levels in cereal species, such as rice. Ascorbate concentrations were significantly increased in germinated brown rice and tissues of 35S-OsGGP plants at the vegetative growth phase. In contrast, foliar ascorbate concentrations were significantly reduced in 35S-OsGGP plants at the reproductive growth phase. This was dependent on homozygosity of the 35S-OsGGP transgene and was associated with a significant reduction in endogenous OsGGP transcript levels, suggesting the occurrence of gene silencing. Foliar ascorbate concentrations were significantly increased in uorfOsGGP mutants, without any changes to OsGGP transcript levels, attributed to alleviated ribosomal stalling on the OsGGP uORF and enhanced translation of the GGP major ORF. Editing the GGP uORF represents an effective transgene-free strategy to increase ascorbate concentrations not only in rice, but other species. Challenging convention, automated imaging revealed that neither the 35S-OsGGP nor the uorfOsGGP plants displayed increased salt tolerance at the vegetative growth phase, despite having elevated ascorbate levels. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells—an accurate predictor of Fe uptake in human digestion—exposed to in vitro digests of null segregant and 35S-OsGGP brown rice and germinated brown rice, suggesting that ascorbate-enriched crops may be able to improve Fe bioavailability in human diets. Grain Fe concentrations were not changed in the uorfOsGGP mutants, indicating that ascorbate may not facilitate the transport of Fe into developing rice grain. Next, this PhD project identified six TaGGP genes in the hexaploid bread wheat genome, each with a highly conserved uORF in the 5’ leader sequence. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. A transcript analysis of the TaGGP genes identified that the TaGGP1 homoeologs were broadly expressed across different tissues and developmental stages and that the TaGGP2 homoeologs were highly expressed in anthers. Finally, transient transformation of the TaGGP coding sequences in Nicotiana benthamiana significantly increased foliar ascorbate concentrations more than five-fold, confirming their activity toward ascorbate biosynthesis in planta. The six TaGGP genes and uORFs identified in this study present an opportunity to fine-tune ascorbate biosynthesis in this important staple crop.