Biomolecular Sciences - Doctoral Theses

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Browsing Biomolecular Sciences - Doctoral Theses by Subject "abiotic stress"

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    Transcriptome and methylome analysis of trembling aspen (Populus tremuloides) under nickel stress.
    (2022-04-28) Czajka, Karolina M.
    Nickel is an essential micronutrient required at low concentrations for adequate plant growth and health. However, excessive amounts of bioavailable nickel ions in the surrounding soil can result in plant toxicity symptoms. Plants have evolved heavy metal tolerance mechanisms to adapt and cope with this abiotic stressor. The main objectives of the present research were to 1) further characterize the P. tremuloides transcriptome 2) compare gene expression dynamics between nickel-resistant and nickel-susceptible P. tremuloides genotypes with Whole Transcriptome (WT) sequencing, 3) determine the effects of different nickel concentrations on P. tremuloides gene expression and, 4) assess global methylation levels in P. tremuloides under nickel stress. Trembling aspen (Populus tremuloides) seedlings treated with varying concentrations of nickel nitrates (150 mg Ni / 1 kg of dry soil, 800 mg / kg, and 1, 600 mg / kg) showed phenotypic segregation of physical toxicity symptoms at the highest nickel dose of 1, 600 mg / kg. This study revealed that a metal transport protein (Potrs038704g29436 – ATOX1-related copper transport) was among the top upregulated genes in resistant genotypes when compared to susceptible plants. Other upregulated genes associated with abiotic stress were identified including a Dirigent Protein 10, GATA transcription factor, Zinc finger protein, Auxin response factor, Bidirectional sugar transporter, and thiamine thiazole synthase. Overall, an upregulation in ribosomal and translation activities was identified as the main response to Ni toxicity in the resistant plants. The results of the dosage analysis suggested that the 800 mg / kg nickel dose is the threshold at which an early abiotic stress response may be triggered as seen by the highly upregulated LEA protein and two calcium binding proteins when compared to water. The cluster of genes that had increased gene expression with increasing nickel dose also had multiple enriched GO terms related to heavy metal and abiotic stress including metal ion transport, antioxidant activity, photosynthesis, and ribosomal activity. Lastly, the initial screen for potential global methylation differences between nickel-resistant genotypes and water showed no significant difference in overall methylation levels. However, the potassium nitrate control for the 1,600 mg / kg dose did show significantl hypomethylation in comparison to the nickel-treated or water control samples. Future experiments could use targetspecific methylation and gene expression assays to investigate the biological significance of the heavy metal stress candidate genes identified in this top-down study in trembling aspen. Understanding the heavy metal tolerance mechanisms and responses used by hardy species like trembling aspen is important for environment bioremediation and maintenance of healthy ecosystems.
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    Unraveling the metabolic networks involved in the utilization of L-glutamine in Pseudomonas fluorescens exposed to nutritional stress
    (2022-06-28) Legendre, Félix
    Sulfur plays an essential role in oxidative homeostasis due to its participation in sulfhydryl groups (SH). A disruption of this vital nutrient is known to promote oxidative stress and activates a plethora of anti-oxidative strategies. Phosphate, a micronutrient that is part of adenosine triphosphate (ATP), the main molecule used as energy and other macromolecules in living cells. The stress response to sulfur and phosphate deficiency in Pseudomonas fluorescens was investigated with emphasis on ROS detoxification, and energy production. Metabolite profiling was performed by High Performance Liquid Chromatography (HPLC), enzymatic analysis was done using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and gene expression assessment of targeted genes was performed with SYBR-Green real-time PCR (qPCR). When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. The up-regulation of isocitrate dehydrogenase (ICDH)-NADP+ dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis. This reaction is aided by the concomitant increase in NAD kinase (NADK) activity. The latter converts NAD+ into NADP+ in the presence of ATP. Additionally, the microbe reprograms its metabolic pathways to produce KG and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminases (GABA-T). Taken together, the data point to a metabolic network involving isocitrate, KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment. Finally, when cultured in low phosphate environments, the microbe can produce ATP via substrate level phosphorylation (SLP), in a mechanism involving the reductive isocitrate dehydrogenase (ICDH-NADH), isocitrate lyase (ICL), malate synthase (MS) as well as phosphoenol pyruvate carboxylase (PEPC), phosphoenol pyruvate synthase (PEPS) and pyruvate phosphate dikinase (PPDK). This metabolic reprogramming ensures the survival of the microbe and reveals the central role metabolism plays in cellular adaptation to abiotic stress.