Plant sciences

Ecological restoration of grassland ecosystems is increasing in scope in Ontario, as a result the demand for genetically appropriate, high-quality seeds of native plants is also increasing. This mixed methods study characterized fitness and seed quality traits using genetic, demographic and growth trial data for a keystone tallgrass prairie species Big bluestem (Andropogon gerardii). To estimate the ploidy levels in Big bluestem, our flow cytometric results indicated an average of 6.32 picograms of nuclear DNA within sixteen populations surveyed showing that hexaploid (6x) cytotypes are dominant in Southern and Central Ontario populations, aside from one 9x occurrence in Norfolk county, ON. Seed quality, measured through germination and viability testing did not change based on whether a population was remnant or restored. Concerningly, our study shows that remnant populations of Big bluestem are at risk of being lost as high quality seed sources likely because of the absence of stewardship and the resulting loss in population fitness. Workshops with prairie restoration practitioners suggest that there is significant vision behind the future of this work in the province, and that an ecosystem wide seed strategy for keystone tallgrass prairie species is a necessary next step to increase the sustainability of seed-based restoration strategies and preserve remnant site genetics.

Author Keywords: Big bluestem (Andropogon gerardii), Ecological Restoration, fitness, polyploidy, practitioner, seed quality

Macrophytes play an important role in aquatic ecosystems, and thus are integral to ecological risk assessments of environmental contaminants. In this dissertation, I address gaps in the assessments of contaminant fate and effects in macrophytes, with focus on glyphosate herbicide use for invasive plant control. First, I evaluated the suitability of Typha as future standard test species to represent emergent macrophytes in risk assessments. I concluded that Typha is ecologically relevant, straight-forward to grow, and its sensitivity can be assessed with various morphological and physiological endpoints. Second, I assessed effects from glyphosate (Roundup WeatherMAX® formulation) spray drift exposure on emergent non-target macrophytes. I performed toxicity tests with five taxa, Phragmites australis, Typha × glauca, Typha latifolia, Ammannia robusta, and Sida hermaphrodita, which in Canada collectively represent invasive, native, and endangered species. I found significant differences in glyphosate sensitivity among genera, and all species' growth was adversely affected at concentrations as low as 0.1% (0.54 g/L), much below the currently used rate (5%, 27 g/L). Third, I assessed the potential for glyphosate accumulation in and release from treated plant tissues. I found that P. australis and T. × glauca accumulate glyphosate following spray treatment, and that accumulated glyphosate can leach out of treated plant tissues upon their submergence in water. Finally, I assessed effects of released glyphosate on non-target macrophytes. I found that P. australis and T. × glauca leachate containing glyphosate residues can stimulate the germination and seedling growth of T. latifolia, but can exert an inhibiting effect on A. robusta, although leachate without glyphosate caused similar responses in both plants. Additionally, I found no negative effects in A. robusta when exposed to glyphosate residues in surface water, or when grown with rhizosphere contact to an invasive plant that was wicked (touched) with glyphosate. My results show that non-target macrophytes can be at risk from glyphosate spray for invasive plant control, but risks can be mitigated through informed ecosystem management activities, such as targeted wick-applications or removing plant litter. Integrating contaminant fate and effect assessments with emergent macrophytes into ecological risk assessments can support the protection of diverse macrophyte communities.

Author Keywords: Ecosystem management, Ecotoxicology, Glyphosate, Herbicide, Invasive plant, Species at risk

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

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

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

In Canada, eastern flowering dogwood (Cornus florida L.) is an endangered tree that occurs only in the Carolinian forest of southern Ontario. Threats to this species include habitat fragmentation and the fungal pathogen dogwood anthracnose (Discula destructiva). I conducted a population genetic analysis using seven nuclear microsatellite markers to determine if fragmented populations are genetically isolated from one another and have low levels of genetic diversity. Genetic comparisons suggest on-going dispersal among sites and relatively high genetic diversity within most sites; however, smaller populations and younger trees were less genetically diverse. I also used linear mixed effects models to assess potential relationships between several ecological variables and the prevalence of dogwood anthracnose. Disease severity was higher in trees on shallow slopes and in larger trees; the latter also had higher likelihood of infection. Insights from this study will be important to incorporate into future management strategies.

Author Keywords: Cornus florida, Discula destructiva, dogwood anthracnose, Eastern flowering dogwood, endangered, population genetics

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

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

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

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