School of BioSciences - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemMetabolic Engineering Strategies to Increase Ascorbate Concentrations in Rice and Wheat( 2020)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.
-
ItemThe physiological effects of artificial light at night on the Australian black field cricket( 2018)The presence of artificial light at night (ALAN) is one of the fastest growing, most pervasive and, until recently, under-appreciated forms of global pollution. Current ALAN levels in urban environments are associated with changes to animal behaviour, dramatic shifts in the timing of life history events, reductions in individual fitness and disrupted physiological processes, including immune function. This thesis explores the physiological effects of ecologically relevant levels of ALAN on a model invertebrate species, the Australian black field cricket, Teleogryllus commodus. In Chapter 1, I reviewed the literature with a particular emphasis on the physiological effects of ALAN, including growth, survival, reproductive success, and immune function. I also speculate as to the potential mechanistic links behind these ALAN induced biological effects. In Chapter 2, I explored experimentally the effects of ecologically relevant levels of ALAN (1, 10 and 100 lux) on life history and fitness traits of the black field cricket. Under controlled laboratory conditions, I reared crickets from egg to adult in an environment with either no ALAN (0 lux) or one of the above dim-ALAN intensities and assessed the consequences of ALAN for growth, survival and reproductive success. I demonstrated that egg hatch, adult survival and reproductive measures were largely unaffected by the presence of ALAN, however juvenile development time was longer and adults were larger when crickets were exposed to any light at night (1, 10 or 100 lux). In Chapter 3, I examined the effects of ALAN (1, 10 and 100 lux) on three key measures of adult immune function (haemocyte concentration, lytic activity, and phenoloxidase activity). The presence of any ALAN (1, 10 or 100 lux) had a clear negative effect on the cellular immune response. Specifically, individuals exposed to any ALAN were unable to increase their haemocyte concentration in response to a stressor challenge. In Chapter 4, I investigated a novel method for the measurement of circulating melatonin in small samples of cricket haemolymph using high-performance liquid chromatography tandem mass spectrometry, with methyl tert-butyl ether (MTBE)/ethyl acetate as an extraction agent. The calibration curve for melatonin was linear in the range of 0.25 and 10 pM (R2 = 0.999), and the limit of detection was 0.25pM. When applied to a set of pilot data from crickets reared under different ALAN environments (0, 1, 10, and 100 lux), the results were however inconclusive, due to small sample sizes. In Chapter 5, I discuss the significance of these findings and their ecological implications. My thesis advances our understanding of the biological ef fects of ALAN for invertebrates, a key taxon contributing to ecological community structure and composition. It is one of the first set of studies to simultaneously investigate multiple traits in the same individuals exposed to lifelong ALAN, and to assess changes in immune function throughout their adult life. Combined, the results presented demonstrate a disruption to physiological processes, and highlight the potential for ALAN to alter the phenology of communities and reduce the overall fitness of individuals.