School of BioSciences - Theses
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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.