Graduate Theses & Dissertations

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
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
Early Responses of Understory Vegetation to Above Canopy Nitrogen Additions in a Jack Pine Stand in Northern Alberta
Abstract Early Responses of Understory Vegetation After One Year of Above Canopy Nitrogen Additions in a Jack Pine Stand in Northern Alberta Nicole Melong Nitrogen (N) emissions are expected to increase in western Canada due to oil and gas extraction operations. An increase in N exposure could potentially impact the surrounding boreal forest, which has adapted and thrived under traditionally low N deposition. The majority of N addition studies on forest ecosystems apply N to the forest floor and often exclude the important interaction of the tree canopy. This research consisted of aerial NH4NO3 spray applications (5, 10, 15, 20, 25 kg N ha-1yr-1) by helicopter to a jack pine (Pinus banksiana Lamb.) stand in the Athabasca Oil Sands Region (AOSR) in northern Alberta, Canada. The main objective was to assess the impacts of elevated N after one year of treatment on the chemistry of understory vegetation, which included vascular plants, terricolous lichens, epiphytic lichens and a terricolous moss species. Changes in vegetation chemistry are expected to be early signs of stress and possible N saturation. Increased N availability is also thought to decrease plant secondary compound production because of a tradeoff that exists between growth and plant defense compounds when resources become available. Approximately 60% of applied N reached the ground vegetation in throughfall (TF) and stemflow (SF). Nitrate was the dominant form of N in TF in all treated plots and organic N (ON) was the dominant form of N in SF in all plots. The terricolous non-vascular species were the only understory vegetation that responded to the N treatments as N concentration increased with increased treatment. Foliar chemistry of the measured epiphytic lichens, vascular species, and jack pine was unaffected by the N treatments. Based on biomass measurements and N concentration increases, the non-vascular terricolous species appear to be assimilating the majority of TF N after one year. Vegetation from the high treatment plot (25 kg N ha-1yr-1) was compared to a jack pine forest receiving ambient high levels of N (21 kg N ha-1yr-1) due to its proximity to Syncrude mining activities. Nitrogen concentrations in plant tissues did not differ between the two sites; however, other elements and compounds differed significantly (Ca, Mg, Al, Fe). After one year of experimental N application, there were no environmental impacts consistent with the original N saturation hypothesis. Author Keywords: Athabasca Oil Sands Region, Canopy Interactions, Jack Pine, Nitrogen, Secondary Chemistry, Understory Vegetation
Cytokinin Oxidase/Dehydrogenase (CKX) Gene Family in Soybeans (Glycine max)
Glycine max (soybean) is an economically important plant species that registers a relatively low yield/seed weight compared to other food and oil seed crops due to higher rates of flower and pod abortion. Alleviation of this abortion rate can be achieved by altering the sink strength of the reproductive organs of soybeans. Cytokinin (CK) plays a fundamental role in promoting growth of sink organ (flowers and seeds) by increasing the assimilate demand. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that catalyses the irreversible breakdown of active CKs and hence reduce the cytokinin content. The current thesis uncovers the members of CKX gene family in soybeans and the natural variations among CKX genes within soybean varieties with different yield characteristics. The identification of null variants of OsCKX2 that resulted in large yield increases by Ashikari et al. (2005) provided a rationale for current thesis. The soybean CKX genes along with the ones from Arabidopsis, Rice and Maize were used to construct a phylogenetic tree. Using comparative phylogeny, protein properties and bioinformatic programs, the potential effect of the identified natural variations on soybean yield was predicted. Five genes among the seventeen soybean CKXs identified, showed polymorphisms. One of the natural variations, A159G, in the gene GmCKX16 occurred close to the active site of the protein and was predicted to affect the activity of enzyme leading to higher accumulation of CKs and hence increased seed weight. Use of such natural variations in marker assisted breeding could lead to the development of higher yielding soybean varieties. Author Keywords: CKX, Cytokinins, Seed weight, Seed Yield, SNPs, Soybeans

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