Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.

Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

Plant form and structure is remarkably diverse encompassing a myriad of shapes and sizes, and is a result of coordinated instances of cell growth and division. This varied form and structure represents adaptive strategies enabling plants to exploit and endure their environment maximizing fitness. Common to both cell growth and division processes, is the requirement of growth material and effectors via the secretory system. The secretory system is a dynamic pathway of organelles connected via vesicular traffic, responsible for protein modification and delivery. Challenges in studying secretion are related to its' dynamic flow between organelles, and the prevalence of redundancy and lethality, which cause difficulties in interpretation and genetic modulation. To further our understanding of the secretory process, we employed a chemical genetic approach to identify small molecule probes of secretion by first screening for inhibitors of cell expansion, followed by a microscopy based screen of GFP marker lines for perturbagens of subcellular structures. Small molecules offer the advantages of temporal and spatial application, have been shown to overcome redundancy and can be varied in concentration to control the severity of effects. We identified twenty-five small molecule probes of secretion and pursued two chemicals with striking properties, polarazine and eroonazole. Polarazine was identified as a cell division inhibitor, affecting phragmoplast structure and function, while eroonazole remodels the endoplasmic reticulum into small vesicles and is implicated in auxin signaling. This approach shows the utility of small molecule probes in furthering our understanding of secretory processes.

HopZ1a is a type III secreted effector (TTSE) protein from Pseudomonas syringae. The goal of this study was to obtain a 3D crystal structure of HopZ1a to provide insight into its biochemical function. The first objective was to obtain HopZ1a protein that was sufficiently abundant and pure for crystallographic studies. Purification conditions were optimized and multiple constructs of HopZ1a were generated using secondary structure prediction programs as well as structural characteristics inherent to TTSEs. Truncations of HopZ1a from the N- and C-terminus led to a soluble, proteolytically resistant construct, HopZ1a66-261. This protein formed granular precipitates in crystallography screens. These conditions will provide the basis for refinement screens aimed at optimizing the HopZ1a crystallization conditions. Overall, the soluble constructs described in this study will provide invaluable tools for future in vitro functional and structural studies of this important family of type III secreted effector proteins.

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

HopZ1a is a type III secreted effector (TTSE) protein from Pseudomonas syringae. The goal of this study was to obtain a 3D crystal structure of HopZ1a to provide insight into its biochemical function. The first objective was to obtain HopZ1a protein that was sufficiently abundant and pure for crystallographic studies. Purification conditions were optimized and multiple constructs of HopZ1a were generated using secondary structure prediction programs as well as structural characteristics inherent to TTSEs. Truncations of HopZ1a from the N- and C-terminus led to a soluble, proteolytically resistant construct, HopZ1a66-261. This protein formed granular precipitates in crystallography screens. These conditions will provide the basis for refinement screens aimed at optimizing the HopZ1a crystallization conditions. Overall, the soluble constructs described in this study will provide invaluable tools for future in vitro functional and structural studies of this important family of type III secreted effector proteins.

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

The transition from cross-fertilization to predominant self-fertilization is considered the most common evolutionary transition in flowering plants. This change in mating system has profound influences on the amounts and patterns of genetic diversity within and among populations, and on key genetic and demographic processes. The main goal of my thesis is to determine the molecular population genetic consequences of this transition in the annual neotropical aquatic plant Eichhornia paniculata (Pontederiaceae) using DNA sequence from individuals sampled from throughout the species’ geographic range. Populations exhibit a wide range of mating patterns associated the evolutionary breakdown of tristyly facilitating specific contrasts between outcrossing and selfing populations. Analysis of molecular variation supported the hypothesis of multiple origins of selfing, including the evolution of two morphologically distinct selfing variants from Central America and the Caribbean. A survey of 10 nuclear loci from 225 individuals sampled from 25 populations demonstrated the joint influence of mating system, population size and demographic bottlenecks in affecting patterns of nucleotide variation. Small selfing populations exhibited significantly lower genetic diversity compared with larger outcrossing and mixed mating populations. There was also evidence for higher population differentiation and a slower decay of linkage disequilibrium in predominately selfing populations from the Caribbean region. Coalescent simulations of the sequence data indicated a bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years ago. To investigate the consequences of transitions from outcrossing to selfing across the genome, I used high-throughput, short-read sequencing to assemble ~27,000 ESTs representing ∼24Mbp of sequence. Characterization of floral transcriptomes from this dataset identified 269 genes associated with floral development, 22 of which were differentially expressed in three independently derived selfing lineages compared to an outcrossing genotype. Evidence for relaxed selection in selfing lineages was obtained from an analysis of a subset of ~8000 orthologous sequences from each genotype, as predicted by theory. Selfing genomes showed an increase in the proportion of nonsynonymous to synonymous changes and relaxation of selection for codon usage bias. My thesis represents the most detailed investigation to date of the molecular population genetic consequences of intraspecific variation in the mating systems of plants.