Graduate Theses & Dissertations


Cytokinins in nematodes
To investigate cytokinins (CKs) in nematodes, CK profiles of a free-living Caenorhabditis elegans and a plant parasitic Heterodera glycines (soybean cyst nematode, SCN) were determined at the egg and larval stages. SCN had higher total CK level than C. elegans; however, CKs in SCN were mostly inactive precursors, whereas C. elegans had more bioactive forms. This is the first study to show that methylthiols are present in nematodes and may affect plant infection. In infectious SCN larvae, methylthiol levels were much higher than in eggs or C. elegans larvae. Furthermore, The CK profiles of SCN-susceptible and resistant Glycine max cultivars at three developmental stages revealed that, regardless of the resistance level, SCN infection caused an increase in root CKs. One resistant cultivar, Pion 93Y05, showed significantly high levels of bioactive N6-isopentenyladenine (iP) in the non-infected roots which indicated a potential role of CKs in soybean resistance to SCN. Author Keywords: Cytokinins, HPLC-MS/MS, Nematode, SCN resistance, Soybean
Characterization of a Zn(II)2Cys6 transcription factor in Ustilago maydis and its role in pathogenesis
Ustilago maydis (D.C.) Corda is a biotrophic pathogen that secretes effectors to establish and maintain a relationship with its host, Zea mays. In this pathosystem, the molecular function of effectors is well-studied, but the regulation of effector gene expression remains largely unknown. This study characterized Zfp1, a putative U. maydis Zn(II)2Cys6 transcription factor, as a modulator of effector gene expression. The amino acid sequence of Zfp1 indicated the presence of a GAL4-like zinc binuclear cluster as well as a fungal specific transcription factor domain. Nuclear localization was confirmed by tagging Zfp1 with enhanced green fluorescent protein. Deletion of zfp1 resulted in attenuated hyphal growth, reduced infection frequency, an arrest in pathogenic development, and decreased anthocyanin production. This phenotype can be attributed to the altered transcript levels of genes encoding predicted and confirmed U. maydis effectors in the zfp1 deletion strain during pathogenic growth. Complementation of zfp1 deletion strain with tin2, an effector involved in anthocyanin induction, suggested this effector is downstream of Zfp1 and its expression is influenced by this transcription factor during in planta growth. When wild-type zfp1 was ectopically inserted in the zfp1 deletion strain, pathogenesis and virulence were partially restored. This, coupled with zfp1 over-expression strains having a similar phenotype as the deletion strains, suggested Zfp1 may interact with other proteins for full function. These findings show that Zfp1, in conjunction with one or more binding partners, contributes to U. maydis pathogenesis, virulence, and anthocyanin production through the regulation of effector gene expression. Author Keywords: effector, pathogenesis, transcription factor, Ustilago maydis, Zea mays, zinc finger
cis-Cytokinins from the tRNA-degradation pathway impact the phenotype and metabolome of Arabidopsis thaliana
Cis-isomers of the cytokinin plant hormone family are thought to have low activity or impact on plant growth and development. Mutants with independent silencing of the pathway leading to cis-CK (cis-cytokinin) were investigated at the phenotype and metabolite levels. Phenotypic deviations were noted in trichome development, fresh weight, rosette diameter, number of non-rosette leaves, shoot height, delayed flowering, flower number, and carotenoids. Exploratory metabolomic analysis detected a number of metabolite features that have been associated with CK, auxin, and ABA (abscisic acid) activity. Evidence from both phenotype and metabolomic analysis support the hypothesis that cis-CK production is biologically important for plant growth and development. Author Keywords: arabidopsis, cytokinin, IPT, metabolmics
Cytokinin biosynthesis, signaling and translocation during the formation of tumors in the Ustilago maydis-Zea mays pathosystem
Cytokinins (CKs) are hormones that promote cell division. During the formation of tumors in the Ustilago maydis-Zea mays pathosystem, the levels of CKs are elevated. Although CK levels are increased, the origins of these CKs have not been determined and it is unclear as to whether they promote the formation of tumors. To determine this, we measured the CK levels, identified CK biosynthetic genes as well as CK signaling genes and measured the transcript levels during pathogenesis. By correlating the transcript levels to the CK levels, our results suggest that increased biosynthesis and signaling of CKs occur in both organisms. The increase in CK biosynthesis by the pathosystem could lead to an increase in CK signaling via CK translocation and promote tumor formation. Taken together, these suggest that CK biosynthesis, signaling and translocation play a significant role during the formation of tumors in the Ustilago maydis-Zea mays pathosystem. Author Keywords: Biosynthesis, Cytokinins, Signaling, Translocation, Ustilago maydis, Zea mays
Effect of Carbon Source and Phytohormones on the in vitro Growth of Euglena Gracilis
Microalgae are a promising source of valuable compounds relevant to biofuels, biomaterials, nutraceuticals as well as animal and human nutriment. Unfortunately, low cell density and slow growth result in reduced economic feasibility. Heterotrophic cell culturing using an organic carbon source in lieu of light has proven to be an effective alternative to photobioreactors; however, further improvement may be possible with the addition of growth promoting phytohormones. In this thesis, growth and endogenous hormone profiles in heterotrophic cultures of Euglena gracilis were evaluated using glucose and ethanol as carbon sources. Cytokinin (CK) and abscisic acid (ABA) were quantified by HPLC-ESI-MS/MS and compared to culture growth dynamics. Exogenous phytohormones treatments were also conducted to determine if they may mitigate nutrient reduction and improve growth. Phytohormones CK and ABA were purified and analyzed at seven points along the growth curve in small scale (250 mL flasks, 100 mL working volume) cultures. Among the key findings was that ethanol cultures undergoing exponential growth, primarily synthesize freebase cytokinins (FBCKs) and methylthiol-cytokinins (MeSCKs), while not producing detectable levels of ABA. In exogenous studies, dry biomass was positively influenced with the addition of exogenous ABA; however, the most notable result revealed the ability of transZ to alleviate nutrient reduction. These findings suggest a communication network in algal culture using FBCKs and MeSCKs, as well as the potential for exogenous hormone supplementation to increase growth rates and overall biomass productivity. Author Keywords: abscisic acid, cytokinin, Euglena gracilis, heterotrophy, phytohormones
Exploring reproduction in wild blue lupine (Lupinus perennis) in comparison to L. polyphyllus and L. albus
Wild lupine (Lupinus perennis) restoration efforts seek to increase and connect populations, using seeds, to facilitate the recovery of endangered butterflys in Ontario. This study observed plant growth and phytohormone levels of L. albus, L. polyphyllus, and L. perennis through stages of seed development, each with varying strategies in growth and reproductive investment. L. polyphyllus is similar to L. perennis in morphology, acting as similar comparable with L. albus, a well-studied annual, as an outgroup comparator. Wild lupines showed a lack of sexual reproductive effort as they did not put as much effort into above ground growth, and few in the population reproduces. They also showed cis-zeatin, a weaker cytokinin, throughout development and had higher amounts of abscisic acid at the end of seed maturity, impacting their ability to develop and germinate. These factors contribute to why wild lupines are difficult to restore using seeds, limiting expansion and challenging restoration. Author Keywords: L. albus, L. perennis, L. polyphyllus, plant physiology, seed development, Wild blue lupine
Involvement of Endogenous Plant Hormones in The Regulatory Network of Fatty Acid Biosynthesis in Soybean Seed
The activities of phytohormones during the reproductive phase have been partially clarified in seed physiology while the biological role of plant hormones in oil accumulation during seed development has been investigated in part only. In this research, fatty acid (FA) contents and hormone profiles, including abscisic acid (ABA) and cytokinins (CKs) of seed samples in four different stages and comparing six soybean varieties have been investigated in order to examine the hypothesis that the endogenous plant hormones play important roles in FA production in soybean seeds. The FA contents increased significantly during this period while the hormone concentrations gradually declined towards the seed physical maturation. However, the interactions between FA contents and hormone profiles were complex and went beyond linear correlations. Hormone metabolism in the earlier stages of seed maturation period demonstrated numerous robust relationships with FA accumulations, as derived from several simple and multiple regression models in the determination of different FA contents. Evaluation of the effects of exogenous ABA and trans-Zeatin (tZ) on FA biosynthesis has revealed that ABA appears to be involved in the accumulations of unsaturated FAs while tZ participated in the synthesis of saturated and unsaturated FAs. Notably, the alterations of FA synthesis differ according to what exogenous hormone concentrations could be used. Author Keywords: Abscisic acid, Cytokinin, Fatty acid, Seed development, Soybean
Regulation of Cytokinins During Kernel Development in High and Low Yielding Oat and Barley Lines
Cytokinins (CKs) are a family of plant phytohormones responsible for many areas of plant growth and development. There are four free base types of CKs found in higher plants, trans-zeatin (tZ), N6-(∆2-isopentenyl)adenine (iP), cis-Zeatin (cZ) and dihydrozeatin (DZ). CK biosynthesis is regulated by adenosine phosphate-isopentenyltransferase (IPT), which is encoded by a multi-gene family in many plant species. There are two types of IPT pathways responsible for CK production, the tRNA pathway and the AMP (ATP/ADP) pathway. The tRNA pathway putatively produces cZ and the latter predominantly produces iP type nucleotides. CKs have long been studied for their role in stress tolerance, signal transduction, and involvement in many areas of plant growth and development. This study focuses on the role of CKs and CK biosynthesis by IPT during kernel development and comparisons of its regulation in high and low yielding barley and oat lines. The sequence of a putative IPT encoding gene in barley and oat was identified by a blast search of other known IPT gene fragments in closely related species. Quantitative Real time PCR results based on primers designed for the putative barley and oat IPT gene revealed changes in expression of IPT during different stages of kernel development, but no significance difference was associated with yield. Correlation of IPT gene expression in barley with cZ CK profiles measured by HPLC-MS/MS, confirms a putative IPT gene is a tRNA- IPT. HPLC-MS/MS results reveal some CK types, such as benzyladenine, are more predominant in higher yielding lines. This suggests different types of CKs play a role in yield production. Future studies on more IPT genes in the barley and oat IPT gene family will outline a more clear representation of the role of IPT in barley kernel development. Author Keywords: Benzyladenine, Cereal grain, Cytokinin, Isopentenyl Transferase, Mass Spectrometry, Real Time PCR
Carbon Exchange along a Natural Gradient of Deciduous Shrub Coverage in the Low-Arctic
Arctic terrestrial ecosystems have experienced substantial structural and compositional changes in response to warming climate in recent decades, especially the expansion of shrub species in Arctic tundra. Climatic and vegetation changes could feedback to the global climate by changing the carbon balance of Arctic tundra. The objective of this thesis was to investigate the influence of increased shrub coverage on carbon exchange processes between atmosphere and the Arctic tundra ecosystem. In this study a space-for-time substitution was used, referred to as a shrub expansion “chronosequence”, with three sites along a natural gradient of deciduous shrub coverage in the Canadian low Arctic. Leaf-level photosynthetic capacity (Amax) of dominating birch shrub Betula glandulosa (Michx.) was significantly higher (P<0.05) at the site where shrubs were more abundant and taller than at the other sites. For all sites, mean Amax in 2014 was significantly lower than in 2013, in part potentially due to differences in precipitation distribution. Bulk soil respiration (RS) rate was significantly higher (P<0.05) at the site with more shrubs compared with the other sites. The differences in RS across sites appeared to be driven by differences in soil physiochemical properties, such as soil nitrogen and soil bulk density rather than soil microclimate factors (e.g. soil temperature, moisture). The three sites were either annual CO2 sources (NEP<0) to the atmosphere or CO2 neutral, with strongest annual CO2 sources (-44.1±7.0 g C m-2) at the site with most shrubs. Overall this study suggests that shrubs tundra carbon balance will change with shrub expansion and that shrub ecosystems in the Arctic currently act as annual carbon sources or neutral to the atmospheric CO2 and further shrub expansion might strengthen the CO2 emissions, causing a positive feedback to the warming climate. Author Keywords: arctic tundra, carbon exchange, climate change, photosynthetic capacity, shrub expansion, soil respiration
Hormonal Algae
Based on an endogenous hormone study, three cytokinin type phytohormones; benzyladenine (BA), trans-zeatin (tZ) and methylthiol trans-zeatin (MeSZ), as well as abscisic acid (ABA) were exogenously added at three concentrations (10-7, 10-6 and 10-5 M) to cultures of Chlorella vulgaris in an attempt to alter growth rate, total lipid and fatty acid yields and fatty acid profile. Growth stimulation was highest at 10-6 M for BA, MeSZ and ABA and 10-5 M for tZ. All treatments caused changes in total lipid and fatty acid content, with BA causing an increase to lipid content. The most significant change in the fatty acid profile was observed with the addition of MeSZ at 10-7 and 10-6 M causing increases of 204% and 457% in linolenic acid respectively above the control. These results are novel and potentially highly impactful, as MeSZ has never been added exogenously to algae and may be used to stimulate overproduction of linolenic acid for pharmaceutical or industrial purposes. Author Keywords: Abscisic Acid, Chlorella vulgaris, Cytokinin, Fatty acid, Linolenic Acid, Methylthiol trans-Zeatin
Bioremoval of copper and nickel on living and non-living Eugelna gracilis
This study assesses the ability of a unicellular protist, Euglena gracilis, to remove Cu and Ni from solution in mono- and bi-metallic systems. Living Euglena cells and non-living Euglena biomass were examined for their capacity to sorb metal ions. Adsorption isotherms were used in batch systems to describe the kinetic and equilibrium characteristics of metal removal. In living systems results indicate that the sorption reaction occurs quickly (<30 min) in both Cu (II) and Ni (II) mono-metallic systems and adsorption follows a pseudo-second order kinetics model for both metals. Sorption capacity and intensity was greater for Cu than Ni (p < 0.05) and were described by the Freundlich model. In bi-metallic systems sorption of both metals appears equivalent. In non-living systems sorption occurred quickly (10-30 min) and both Cu and Ni equilibrium uptake increased with a concurrent increase of initial metal concentrations. The pseudo-first-order model was applied to the kinetic data and the Langmuir and Freundlich models effectively described single-metal systems. The biosorption capacity of Cu (II) and) was 3x times greater than that of Ni (II). Sorption of one metal in the presence of relatively high concentrations of the other metal was supressed. Generally, it was found that living Euglena remove Cu and Ni more efficiently than non-living Euglena biomass in both mono- and bi-metallic systems. It is anticipated that this work should contribute to the identification of baseline uptake parameters and capacities for Cu and Ni by Euglena as well as to the increasing amount of research investigating sustainable bioremediation. Author Keywords: accumulation, biosorption, Cu, Euglena gracilis, kinetics, Ni
Fungi and Cytokinins
Cytokinin biosynthesis in organisms aside from plant species has often been viewed as a byproduct of tRNA degradation. Recent evidence suggests that these tRNA degradation products may actually have a role in the development of these organisms, particularly fungi. This thesis examines the importance of cytokinins, a group of phytohormones involved in plant cell division and differentiation as well as the phytohormone abscisic acid, involved in plant response to environmental factors, and their presence and role in fungi. An initial survey was conducted on 20 temperate forest fungi of differing nutritional modes. Using HPLC-ESI MS/MS, cytokinin and abscisic acid were detected in all fungi regardless of their mode of nutrition or phylogeny. The detection of the same seven CKs across all fungi suggested the existence of a common CK biosynthetic pathway and dominance of the tRNA pathway in fungi. Further, the corn smut fungus Ustilago maydis is capable of producing CKs separate from its host and different U. maydis strains induce disease symptoms of differing severity. To determine if CK production during infection alters disease development a disease time course was conducted on cob tissue infected with U. maydis dikaryotic and solopathogenic strains. Dramatic changes in phytohormones including an increase in ABA followed by increases in cisZCKs were detected in tumour tissue particularity in the more virulent dikaryon infection, suggesting a role for CKs in strain virulence. Mining of the U. maydis genome identified a sole tRNA-isopentenyltransferase, a key enzyme in CK biosynthesis. Targeted gene deletion mutants were created in U. maydis which halted U. maydis CK production and decreased pathogenesis and virulence in seedling and cob infections. CK and ABA profiling carried out during disease development found that key changes in these hormones were not found in deletion mutant infections and cob tumour development was severely impaired. These findings suggested that U. maydis CK production is necessary for tumour development in this pathosystem. The research presented in this thesis highlights the importance of fungal CKs, outlines the dominant CK pathway in fungi, identifies a key enzyme in U. maydis CK biosynthesis and reveals the necessity of CK production by U. maydis in the development of cob tumours. Author Keywords: abscisic acid, cytokinins, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, tRNA degradation pathway, Ustilago maydis, Zea mays


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