With the goal of creating faster and more efficient methods of generating unique Ac/Ds insertional mutants in a population of Fragaria vesca, various methods of seed germination, kanamycin screening, and the effects of varying nitrogen fertilization on diploid strawberry have been examined. Seed germination was improved to 42% in B5 liquid medium compared to _ on MS solid medium. Kanamycin screening during germination was most effective in liquid B5 medium as well. A readily discernable phonotypic difference between sensitive (necrotic radical) and resistant (branched roots) seedlings was observed in the B5 liquid medium and the frequency of escapes was reduced from __ on solid MS to __ in liquid B5. Although there were few phenotypic differences due to nitrogen application over the tested treatments (25-300 ppm) runner initiation was suppressed and chlorophyll was increased in the high (300 ppm) nitrogen treatment. There was limited evidence to suggest an increased rate of transposition in the high (300 ppm nitrogen) treatment level compared to those plants receiving lower levels of nitrogen. The selection efficiency and greater germination of the B5 liquid medium over MS medium would be expected to reduce the cost of screening thousands of seedlings because of the need for fewer disposables and medium transfers during the 5 week germination process. The use of B5 liquid medium, as well as treating plants with high levels of nitrogen (300 ppm), may be facilitate high throughput production of transposon tagged mutants in a population of F. vesca. / Master of Science

Activation-tagging is a functional genomics technique where strong enhancers are inserted randomly into target genomes to over-activate endogenous genes. Phenotypes of interest can be selected for investigation of genetic factors contributing to the mutant phenotype. From initial screens of a population of activation-tagged potato, a mutant with chocolate-coloured tuber skin has been identified. In this thesis, a novel sequence capture method for identifying T-DNA loci in activation tagged potato was used to characterize chocolate’s single T-DNA insertion locus. Transcriptome analysis of tuber periderm tissue was used to identify major processes occurring in the chocolate mutant. Our data suggest activation of a chitin-binding receptor-like kinase located 65 kb from T-DNA insert may cause activation of immune signaling pathways in chocolate. The present work explores a putative model of transcriptional and cellular responses involved in gain-of-function immune receptor activation. Selectively, these findings illustrate the periderm tissue as an important area of defense charged against biotic and abiotic stresses. Periderm development and anatomy are highly important for tuber storage. Further characterization of potato tuber periderm may contribute knowledge to model periderm systems and have implications for molecular breeding strategies to improve tuber storage quality. / Thesis (Master, Biology) -- Queen's University, 2012-09-27 11:45:16.478

Our understanding of tree growth and development has increased substantially in the last few decades and is expected to increase much more as we fully exploit newly developed genomic tools. A major milestone in tree genomics was the sequencing of the entire genome of Populus trichocarpa, and the realization that we understand the function of very few of the 45,000 predicted genes in this genome. To advance our knowledge of gene function in Populus, we have created the largest population of mutant poplars to date which will enable us to link altered phenotypes with genes that are responsible. This thesis describes this mutant population, provides preliminary results on the complexity of mutants identified and examines one distinct mutant called shriveled leaf. These results clearly demonstrate the power of this population for gene function analysis and reveal that this population will be a valuable genomic resource for tree biotechnology for many years to come. / Thesis (Master, Biology) -- Queen's University, 2008-01-31 16:09:03.458

Plant-wide communication between tissues and cells is organized, in part, by a suite of compounds called hormones. I have chosen to focus on the effects of one plant hormone, ethylene; how its synthesis is controlled and how its perception is mediated to differentially control cell development and response to pathogens. In the production of ethylene, one level of control is by modulating the levels of the immediate precursor to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). I characterize here a plant encoded gene homologous to bacterial ACC Deaminases, AtACD1, and show through up- and down-regulation of the gene that it can modulate the plants sensitivity to exogenous ACC. Once ethylene is produced, it is sensed in Arabidopsis thaliana by a family of 5 receptors. I show that ETR2 in Arabidopsis is responsible for modulation of the microtubule cytoskeleton assembly as loss-of –function mutations to this gene cause randomized microtubule assembly in trichomes and increase sensitivity to microtubule depolymerising drugs in root hairs. In studies of plant:pathogen interactions, ethylene is a central signaling agent required for plant resistance. While it has been shown that etr1 mutants show increased susceptibility to fungal pathogens, exogenous ethylene has also been shown to speed the progress of pathogenesis. Using Fumonisin B1 (FB1) to induce cell death I show that etr1-1 has accelerated cell death while ein4-1 has a reduced rate of necrosis. Further to this, mutations to the other three ethylene receptors do not have any effect on the rate of cell death. My interest in cell development led to the characterization of an activation tagged Populus tremula x P. alba line with increased trichome initiation. The gene responsible for these phenotypes was identified as PtMYB186, which also affected growth rate, transpiration rate, photosynthetic capacity, and resistance to the Tussock moth larvae. Together these studies provide a new framework for our understanding of how the ethylene signal is modulated in plants and the controls behind cellular development. This knowledge will help reconcile studies which show that ethylene has different effects on plant development and provide new avenues of research into trichome development. / Thesis (Ph.D, Biology) -- Queen's University, 2009-01-13 10:08:03.605

Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination. / Ph. D.

BT2 is an Arabidopsis thaliana protein with N-terminal BTB, central TAZ and a C-terminal calmodulin binding domain and associates with Cullin3 to form an E3 ubiquitin ligase. We have shown previously that BT2 regulates telomerase activity in mature vegetative organs and controls a variety of hormone, stress and metabolic responses in Arabidopsis thaliana. Loss of BT2 results in plants that are hypersensitive to inhibition of germination by ABA and sugars. Conversely, overexpression of BT2 results in resistance to ABA and sugars, suggesting that BT2 is a negative regulator of ABA and sugar responses. Here, we report the roles of BT2-interacting partners GTE9 and GTE11, bromodomain and extraterminal-domain proteins of Global Transcription Factor Group E, in BT2-mediated responses to sugars and hormones. Loss-of-function mutants gte9-1 and gte11-1 phenocopy the bt2-1-null mutant responses; germination in all three mutants is hypersensitive to inhibition by glucose and ABA. Loss of either GTE9 or GTE11 in a BT2 over-expressing background blocks resistance to sugars and ABA, indicating that both GTE9 and GTE11 are required for BT2 function. Additionally, loss of GTE9 or GTE11, similar to loss of BT2, suppresses transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. The suppressed phenotype was accompanied by decreased transcription and hypermethylation of the 35S enhancers in the activation-tagged lines. We showed that BT2 and GTE9 co-immunoprecipitate and physically interact in vivo to mediate diverse responses to biotic and abiotic signals and 35S enhancer activity. Our working model is that the GTE9 and GTE11 function as chromatin adaptors that localize the BT2-CULLIN3 E3 ubiquitin ligase complex to acetylated chromatin on transcriptionally competent promoters in response to calcium signals detected by BT2’s calmodulin-binding domain.

As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells. / Master of Science