This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

We previously described BT2, a BTB/POZ domain containing protein, as an activator of telomerase in Arabidopsis thaliana. In the current study, I present evidence of its interesting roles in mediating multiple hormone, stress and metabolic responses in plants. Steady-state expression of BT2 mRNA was regulated diurnally and was under the control of circadian clock, with a maximum expression in the dark. BT2 mRNA was responsive to nutrient status and to multiple biotic and abiotic stress signals. Using bt2 loss-of-function and BT2 over-expressing lines, I show that BT2 suppresses sugar and ABA-mediated responses during germination. BT2 is also essential for transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. Loss of BT2 in several well-characterized 35S enhancer activation-tagged lines such as yucca1d, pap1d, jaw1d etc., resulted in suppression of the activation phenotypes. The suppression of the phenotypes was due to decreased transcription of the activation-tagged genes. I further demonstrate that BT2 genetically interacts with CULLIN3. I propose that BT2 and CULLIN3 are components of a ubiquitin ligase complex. Together with associated proteins BET9 and BET10, the BT2 complex is required for CaMV 35S enhancer-mediated activation of gene expression and may regulate expression of target genes involved in multiple responses to fluctuating biotic and abiotic conditions. I also found that BT2 protein levels are tightly regulated in plants. BT2 protein was primarily localized in the nucleus and was developmentally regulated. BT2 turn-over was regulated in part by the 26S-proteosome, and rare codons present in its open reading frame affected BT2 protein accumulation. In addition to BT2, its orthologs, BT1, BT3, BT4 and BT5, also responded to light, clock and nutrients, with some differences. Moreover, BT1, BT3 and BT4 were also required for 35S enhancer-mediated activation of gene expression. I propose that BT family proteins assemble into multi-protein complexes to mediate multiple responses to changing environmental and nutritional conditions.

Crop lands around the world are becoming more salt-affected due to natural processes and agricultural practices. Due to this increase of salinization, acquisition of saline tolerant germplasm for breeding purposes is becoming a priority. Although cultivated sunflower is classified as a moderately salt tolerant crop, highly tolerant germplasm may be of value. The goal of this study was to screen Helianthus spp. in order to determine the salt tolerance of different genotypes. To accomplish this goal, a novel method of rapid screening was developed. Screening for tolerance at initial growth stages was accomplished by germinating seeds in varying concentrations of NaCl solution in petri dishes. Radicle lengths were measured as an indicator of tolerance. This method identified genotypes that are more tolerant than others during germination. Greenhouse trials were also conducted to ascertain morphological measurements during vegetative stages. Two field locations were chosen to screen germplasm for tolerance through physiological maturity; College Station, TX with low salt concentrations and Pecos, TX with high concentrations of salt in the soil and water. Vegetative growth measurements showed a significant genotype by environment interaction. Due to insect infestation in both locations, yields could not be accurately measured and thus compared between sites in 2010. Yields between locations in 2011 showed significant differences and identified germplasm more suited for cropping in salt affected soil. Seed oil content was determined with Fourier Transform Near-Infrared Spectroscopy. Seed oil content was not significantly different between locations, but was highly significant between genotypes. These screenings identified genotypes that are more salt tolerant than others.

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

No description available.

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

Lake sturgeon, recently recommended to be listed as an endangered species under the Species at Risk Act, inhabit the various impoundments of the Winnipeg River system. Downstream passage through hydroelectric generating stations represents one of the major data gaps in our understanding of how hydroelectric development may be impacting lake sturgeon populations. Acoustic telemetry was used to investigate coarse-scale movements of juveniles, sub-adult and adults throughout the Slave Falls Reservoir, a 10 km long Winnipeg River impoundment, to assess patterns of downstream passage susceptibility and investigate fine-scale movements in the vicinity of the Slave Falls Generating Station. Movements of juveniles and sub-adults were generally restricted to areas of interconnected deep-water habitat, with movements through the shallow river narrows that sub-divide the Slave Falls Reservoir being rare. Adults did move through these narrows, albeit infrequently. Juveniles and sub-adults tagged in the lowermost section of the reservoir, as well as several adults tagged throughout the reservoir, were found to periodically utilize habitat immediately upstream of Slave Falls, where they would be susceptible to entrainment. Mean entrainment rates were estimated at 3.1% per year for adults tagged throughout the reservoir, and 17.9% per year for sub-adults tagged in the lowermost section of the reservoir. Fine-scale movement tracking revealed that three of eleven observed downstream passage events occurred via bottom-draw regulating gates, while another four events were also reasoned to have occurred via this route. The routes of the remaining four could not be determined. Eight of the eleven downstream passage events observed in this study were survived. While the survival of the remaining three fish could not be confirmed, it is highly likely that they also survived. Length-at-age analysis, supported by genetic methods, revealed that 23 of 151 (15.2%) of the lake sturgeon between 525 and 750 mm (fork length) captured in the 6 km stretch of river downstream of Slave Falls were fast-growing outliers, reasoned to have passed downstream through the Slave Falls Generating Station.

Tall fescue is the most common cool-season grass in the eastern USA, with broad economic and ecological importance to the region. Tall fescue is known to associate with a fungal endophyte, Neotyphodium coenophialum, whose presence can decrease biotic and abiotic stress experienced by the plant. This thesis evaluates the response of tall fescue and the fungal endophyte symbiosis to predicted climate change. I participated in two multi-factor climate change projects where I investigated the response of tall fescue tissue chemistry and growth to various climate change factors. Endophyte-infected (E+) tall fescue had decreased alkaloid production under elevated CO2 but increased alkaloid production under elevated temperatures. Significant differences between E+ and E- (endophyte-free) tall fescue tissue chemistry were also found, suggesting the endophyte interacts with the plant response to abiotic stress. Although several studies have reported benefits of endophyte infection for tall fescue growing under drought stress, my research found no differences between E+ and E- total growth and surprisingly showed increased mortality of E+ individuals under elevated temperature. Taken together, my research indicates that this grass-fungal relationship will respond to climate change, and may produce dramatic and unforeseen results that question the widely believed mutualistic nature of the symbiosis.

Faba bean (Vicia faba L.) is a cool season crop that uses symbiotic biological nitrogen fixation to obtain atmospheric nitrogen (N), a limiting macronutrient, for growth and maintenance of the plant. Most cool season legumes like faba bean transport N from the nodules as amides, which are metabolized in destination tissues. Ureide metabolism is a catabolic process that produces N rich compounds from purine rings. Many warm season legumes such as soybean and common bean produce ureides (allantoin and allantoate) in their root nodules and then use these molecules to transport fixed nitrogen from root to shoot. Non-ureide exporting plants such as faba bean also produce ureides in normal purine recycling whereby these compounds may play a role in response to abiotic stress. This research aims to examine possible differences in ureide metabolism across genotypes and to assess the role of ureides in response to water limitation. In field grown faba bean, total ureides were found in highest concentrations in leaf tissue, followed by reproductive parts, stems, and nodules, but were not found to differ significantly among genotypes. Ureide concentrations varied throughout the growing season, decreasing over time as the plants reached physiological maturity. A water limitation experiment of faba bean grown in a controlled environment showed that faba bean accumulated the ureides allantoin and allantoate after six to eight days of water limitation when all data were pooled. However, no consistent trend was observed comparing results by genotype, and inoculated versus non-inoculated plants. Overall, results indicate that faba bean likely does not use ureides to transport symbiotically fixed N and that ureide accumulation in field grown plants is most likely in response to abiotic stress or remobilization of purine N from senescing tissues.

Lake sturgeon, recently recommended to be listed as an endangered species under the Species at Risk Act, inhabit the various impoundments of the Winnipeg River system. Downstream passage through hydroelectric generating stations represents one of the major data gaps in our understanding of how hydroelectric development may be impacting lake sturgeon populations. Acoustic telemetry was used to investigate coarse-scale movements of juveniles, sub-adult and adults throughout the Slave Falls Reservoir, a 10 km long Winnipeg River impoundment, to assess patterns of downstream passage susceptibility and investigate fine-scale movements in the vicinity of the Slave Falls Generating Station. Movements of juveniles and sub-adults were generally restricted to areas of interconnected deep-water habitat, with movements through the shallow river narrows that sub-divide the Slave Falls Reservoir being rare. Adults did move through these narrows, albeit infrequently. Juveniles and sub-adults tagged in the lowermost section of the reservoir, as well as several adults tagged throughout the reservoir, were found to periodically utilize habitat immediately upstream of Slave Falls, where they would be susceptible to entrainment. Mean entrainment rates were estimated at 3.1% per year for adults tagged throughout the reservoir, and 17.9% per year for sub-adults tagged in the lowermost section of the reservoir. Fine-scale movement tracking revealed that three of eleven observed downstream passage events occurred via bottom-draw regulating gates, while another four events were also reasoned to have occurred via this route. The routes of the remaining four could not be determined. Eight of the eleven downstream passage events observed in this study were survived. While the survival of the remaining three fish could not be confirmed, it is highly likely that they also survived. Length-at-age analysis, supported by genetic methods, revealed that 23 of 151 (15.2%) of the lake sturgeon between 525 and 750 mm (fork length) captured in the 6 km stretch of river downstream of Slave Falls were fast-growing outliers, reasoned to have passed downstream through the Slave Falls Generating Station.