Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1°C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea’s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.

Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1°C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea’s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.

Doctor of Philosophy / Horticulture, Forestry, and Recreation Resources / Sunghun Park / Abiotic stresses, including chilling and drought stresses, are considered to be major limiting factors for growth and yield of agricultural and horticultural crops. One of the inevitable consequences of abiotic stresses is the accumulation of reactive oxygen species (ROS) in plants. ROS can either act as an alarm signal to induce the defense pathway when kept at a low level or cause oxidative damage to various cellular components when increased to a phytotoxic level. Glutaredoxins (GRXs) are members of ROS scavenging system that can maintain the cell redox homeostasis by using the reducing power of glutathione. In this research, we characterized the roles of GRXs in protecting tomato (Solanum lycopersicum) from chilling stresses and rice (Oryza sativa L.) from drought stresses. Our results indicated that ectopic expression of an Arabidopsis gene AtGRXS17 in tomato could enhance the chilling tolerance by increasing antioxidant enzyme activities and reducing H₂O₂ accumulation to ameliorate oxidative damage to cell membranes and photosystems. Furthermore, AtGRXS17-expressing tomato plants had increased accumulation of soluble sugars to protect plant cells from dehydration stress. In rice, silenced expression of a rice glutaredoxin gene OsGRXS17 was used as a reverse-genetic approach to elucidate the roles of OsGRXS17 in drought stress tolerance. Our results showed that silenced expression of OsGRXS17 conferred improved tolerance to drought stress in rice. ABA-mediated stomatal closure is an important protection mechanism that plants adapt to a drought stress conditions, and H2O2 acts as secondary messenger in ABA signaling to induce the stomatal closure. Silenced expression of OsGRXS17 gave rise to H₂O₂ accumulation in the guard cells and promoted ABA-mediated stomatal closure, resulting in reduced water loss, higher relative water content, and consequently enhanced drought tolerance in rice. This research provides a new perspective on the functions of GRXs in chilling and drought stress tolerance of tomato and rice, and an important genetic engineering approach to improve chilling and drought stress tolerance for other crop species.

Doctor of Philosophy / Department of Agronomy / Clenton E. Owensby / Most rangelands are harvested at some point during the year and removal of plant leaf area and biomass alters a host of ecosystem processes including gas exchange. An experiment was conducted in 2005 and 2006 to study the effects of clipping tallgrass prairie at different dates on water vapor and CO2 fluxes. A portable, non-steady-state chamber was designed to measure CO2 and water vapor fluxes from small plots in less than 40 s. A combination of sunlit and shaded readings allowed measurements of net carbon exchange (NCE) and ecosystem respiration (RE); by summing NCE and RE, gross canopy photosynthesis (GCP) was calculated. Throughout the two-year study, the chamber had a minimal effect on microclimate, i.e., average chamber temperature increased 2.9° C, while chamber pressure increased only 0.3 Pa during measurements, and photosynthetically active radiation attenuation was 10%. The immediate effect of all clipping treatments was a loss of leaf area that led to reductions in GCP, NCE, and RE and in most cases decreased water vapor flux. Further patterns of carbon flux were governed by the amount of water stress during canopy development, while water vapor flux rates varied with water availability. Canopies that developed during periods of low water stress quickly increased carbon flux rates following precipitation after a mid-season drought. However, flux rates of canopies, which developed during the mid-season drought, responded considerably slower to subsequent water availability. A separate experiment was conducted from June-October of 2006 to estimate GCP, leaf area index (LAI), and total aboveground biomass with a hyperspectral radiometer. Indices such as the Normalized Difference Vegetation Index and the Simple Ratio were used to estimate LAI and biomass had poor correlations with measured values. However, GCP was significantly correlated to all six indices derived in this study. While GCP measured from June-October was significantly correlated with all indices, removal of the senesced canopy scans recorded during October greatly increased the relationship.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Channa B. Rajashekar / Studies were conducted to examine the role of antioxidants in adaptation of lettuce (Lactuca sativa L.) to unprotected environmental conditions and various environmental stresses. Antioxidants, in addition to being a plant defense mechanism, are phytochemicals that provide significant health-promoting and nutritive value in human diet. Various approaches involving mild environmental stresses and cultural and management practices have been used to enhance the quality of this commonly consumed leafy vegetable by improving its phytochemical and health-promoting attributes. Lettuce plants grown in protective environments adapt to unprotective environmental conditions by activating antioxidant genes such as phenylalanine ammonia-lyase (PAL), gamma-tocopherol methyl transferase (gamma-TMT), and L-galactose dehydrogenase (L-GalDH) involved in the biosynthesis of phenolic compounds,alpha-tocopherol and ascorbic acid, respectively. Mild environmental stresses such as heat shock, chilling, water stress and high light also activate these genes leading to the accumulation of secondary metabolites and phytochemicals without any adverse effect on biomass accumulation. The phytochemicals included chlorogenic acid, chicoric acid, caffeic acid, quercetin-3-O-glucoside and luteolin-7-O-glucoside. However, under field conditions, application of drought stress did not produce consistent results with regard to the phytochemical composition of lettuce. Plants grown in open field have higher phenolic content and higher antioxidant capacity than those grown in high tunnel. However, these plants also had less biomass accumulation. Many factors such as plant age, variety, fertilization, transplanting shock affected the phytochemical composition of lettuce. The red leaf variety 'Red Sails' had higher antioxidant capacity than 'Baronet' and younger plants had the highest phytochemical content. With regard to the management practices, there was no significant difference in phytochemical composition between organically and conventionally managed crops. Also, low fertility favored the accumulation of phytochemicals and increased the antioxidant capacity. Thus, the results show that mild environmental stresses along with appropriate cultural and management practices can enhance the quality of lettuce by improving their phytochemical composition.

My master's work was done within the context of the PhytoVaLix project, a research project that brings together researchers from the Université de Montréal and private companies. The goal of this project is to develop a technology for the phytotreatment of leachate from engineered landfill sites using willows to replace conventional techniques. A filtering plantation of miyabeana willow (Salix miyabeana) is currently in place on the experimental site. The willows are watered with leachate so that they use ammoniacal nitrogen, the main pollutant, for their growth. In my study, I focused on the morphological and physiological responses developed by four willow species when subjected to various treatments combining flooding and nitrogen overfertilization. The study aimed to compare the potential of these species for nitrogenous water phytoremediation as well as two leachate application methods. Responses to flooding and overfertilization with nitrogen varied and reveal much about the strategies adopted by each species. The biomass of S. miyabeana, S. amygdaloides and S. nigra was not impacted by the leachate treatments, while S. bebbiana suffered greatly from the flooding periods. The native species S. nigra and S. amygdaloides positively stood out in their ability to remove nitrogen from leachate (>75 kg/ha). It would be relevant to continue research on these two with the aim of using them in projects where leachate phytotreatment and biodiversity go hand in hand. / Mon travail de maitrise a été réalisé dans le cadre du projet PhytoVaLix, un projet de recherche qui réunit des chercheurs de l'Université de Montréal et des entreprises privées. Le but de ce projet est de développer une technologie de phytotraitement de lixiviat provenant de lieux d’enfouissement technique à l’aide de saules pour remplacer les techniques conventionnelles. Une plantation filtrante de saule miyabeana (Salix miyabeana) est présentement en place sur le site expérimental. Les saules y sont arrosés avec du lixiviat afin qu’ils utilisent l’azote ammoniacal, le polluant principal, pour leur croissance. Dans mon étude, je me suis intéressée aux réponses morphologiques et physiologiques développées par quatre espèces de saule lorsqu’elles sont soumises à divers traitements combinant l’inondation et la surfertilisation à l’azote. L’étude avait comme objectif de comparer le potentiel de ces espèces pour la phytoremédiation d’eau azotée et de deux méthodes d’application du lixiviat. Les réponses à l’inondation et à la surfertilisation à l’azote furent variées et en révèlent beaucoup sur les stratégies adoptées par chacune des espèces. La biomasse de S. miyabeana, S. amygdaloides et S. nigra ne fut pas impactée par les traitements de lixiviat, alors que S. bebbiana a grandement souffert des périodes d’inondation. Les espèces indigènes S. nigra et S. amygdaloides se sont positivement démarqués quant à leur capacité à retirer l’azote du lixiviat (>75 kg/ha). Il serait pertinent de continuer à faire des recherches sur celles-ci dans le but de les utiliser dans des projets où le phytotraitement du lixiviat et la biodiversité vont de pair.

Master of Science / Department of Agronomy / P. V. Vara Prasad / In many countries producers have been growing varieties of hard white winter (HWW) wheat since decades. The cause of concern is most varieties of HWW wheat are susceptible to pre-harvest sprouting (PHS) which affects grain quality. Environmental conditions like high humidity, precipitation, heavy dew and hormonal activity at physiological maturity stimulate PHS in HWW. To alleviate these conditions research was carried out at KSU. KS01HW163-4, a sprouting tolerance line was crossed with Heyne, a sprout susceptible cultivar. A total of 224 doubled haploid (DH) lines thus produced were phenotyped in the present study through experiments conducted in controlled environments. The objectives of this research were to (i) characterize and phenotype the doubled haploid lines for PHS in controlled environments; (ii) understand the impact of growth environment (high temperature and/or drought) and; (iii) impact of exogenous application of growth hormones on tolerance to pre-harvest sprouting in the parental lines of the doubled haploid population. The phenotypic data collected from this research will be ultimately combined with the genotypic data to identify DNA markers related to PHS tolerance and provide DNA markers for marker assisted selection. Based on my results of the germination percentages from the 224 DH lines, the population was distributed as susceptible, and tolerant to PHS showing a bimodal distribution and X[superscript]2 analysis indicating a complimentary gene action. From the study of the influence of environmental factors on PHS, my results confirmed a definite influence of stress on sprouting. Under optimum temperature (OT), KS01HS163-4 was tolerant to PHS, but at HT and/or drought it became susceptible to PHS. Growth under stressed conditions changed the tolerance levels to PHS. Seed dry-weight, and harvest index were also influenced negatively due to stress. Therefore multi-location tests must be conducted with variable environments to test the stability of a variety to PHS. From the study of the influence of phytohormones on PHS, the results suggest that tolerance to sprouting was seen in seeds from plants sprayed with abscisic acid (ABA) and paclobutrazol (GA-inhibitor) treatments where as those from gibberellic acid (GA) treatment showed susceptibility to sprouting.

Master of Science / Department of Agronomy / Robert M. Aiken / Mary B. Kirkham / Transpiration efficiency (TE) is defined as total biomass produced per unit of water transpired. Improvement of TE means maximizing crop production per unit of water used. The objectives of the study were to examine, at the leaf level and the whole plant level, the variation in TE for sorghum [Sorghum bicolor (L.) Moench] accessions, previously screened for TE and to test physiological mechanisms that may account for differences in TE. Three field studies and two mini-lysimeter studies (one done in pots under greenhouse conditions and one done in pots in the field) were conducted with eight accessions. Instantaneous measurements of assimilation (A), stomatal conductance (gs), and transpiration by gas exchange provided measures of the transpiration efficiency at the leaf level. Growth observations and soil water balance in field plots quantified components of whole-plant TE. Growth and development measurements showed significant difference, explaining the existence of photoperiod sensitivity among the sorghum genotypes. Assimilation (A), stomatal conductance (gs), and maximum quantum efficiency of photosystem II (Fv/Fm) were consistently greater for accession PI533946 (from India) and greater for accession PI295121 (from Australia) in both field and the field-pot studies (p<0.05). Internal carbon dioxide (Ci), an indicator of intrinsic transpiration efficiency, differed among lines under field conditions (p<0.05). Leaf relative water content (RWC), measured in the greenhouse, and did not differ among the eight accessions. No consistent differences in biomass and water use were detected among lines under field conditions. In conclusion, developing reliable selection indices for TE will require a greater understanding of whole-plant physiological processes to utilize the differences in TE observed at the leaf level.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

Le but de cette étude est de comprendre l’effet d’une irrigation par les eaux usées et /ou de la fertilisation par les engrais chimiques sur la productivité aérienne et souterraine d’une plantation de saule Salix miyabeana SX67 en CICR dans un contexte de filtre végétal. Nous avons d’une part évalué l’impact de diverses doses d’eau usées et/ou de la fertilisation minérale sur les rendements en biomasse ligneuse d’une culture de saules au cours d’un cycle de croissance de deux ans. D’autre part et pour la même période nous avons comparé le développement racinaire (biomasse, morphologie et distribution dans le sol) suite aux divers traitements. Les résultats ont montré qu’au terme de deux ans de croissance, les traitements par les eaux usées aussi bien que celle par les engrais a permis l’augmentation des rendements de la biomasse aérienne de notre culture de saules avec un effet plus prononcé suite au traitements des eaux usées qu’à celui du fertilisant chimique. Nous avons mesuré des productivités en biomasse aussi élevées que 39,4 Mg ha-1 et 54,7 Mg ha-1 et ce pour les parcelles qui ont reçu la plus grande quantité d'eaux usées, respectivement pour les saules non fertilisé et fertilisé (D3-NF et D3-F). La majeure partie du système racinaire était en superficie avec 92-96% des racines (racine fine et racine grosse) concentrées dans les premiers 40 cm de sol et nous avons trouvé que la biomasse des racines fines était comprise entre 1,01 et 1,99 Mg ha-1. Généralement la fertilisation chimique n’a pas eu d’effet sur les rendements en biomasse des racines totales et/ou fines. Bien que l’irrigation par les eaux usées ait entraîné une réduction statistiquement significative de la biomasse racinaire, néanmoins cette réduction n'était pas linéaire (avec une réduction de la biomasse de D0 à D1, une augmentation de D1 à D2 pour réduire de nouveau de D2 à D3). Cette tendance porte à penser qu'au-delà d'une certaine quantité d'eau et de nutriments (suite à l’irrigation par les eaux usées), le développement du système racinaire des saules est affecté négativement, et bien que la biomasse aérienne soit restée élevée sous le traitement D3, nous pensons que le développement de la plante a été quelque peu déséquilibré. Aucun changement significatif n'a été constaté dans les traits morphologiques liés à l'irrigation par les eaux usées. / The aim of this study was to understand the effect of both wastewater and/or mineral fertilisation on above- and belowground biomass of a Salix miyabeana SX67 willow SRC in a filter vegetation context. We firstly assessed the impact of various doses of wastewater and/or mineral fertilization on biomass yield after two season growth. On the other hand and during the same period we estimated root production and assessed vertical root distribution and roots morphology in response to various treatments doses. The results showed that after two years of growth both mineral fertilization and wastewater sewage fertilization enhanced willow aboveground biomass yields, with a more accurate effect due to wastewater irrigation than the mineral fertilization. We recorded high biomass yields such as 39.4 Mg ha-1 and 54.7 Mg ha-1 respectively for unfertilized and fertilized plants plots which benefits with the largest amount of wastewater (D3-NF and D3-F). We found that most of the roots were contained in top soil layers with 92-96% of the total roots (including fine and coarse roots) concentrated within the first 40 cm of soil depth, fine root biomass ranged between 1.01 and 1, 99 Mg ha-1. Neither mineral fertilization nor wastewater sewage irrigation showed an effect on total or fine roots biomass yields. Although we found a statistically significant decrease prior to wastewater treatments, this reduction was not linear (with a decrease of the biomass from D0 to D1, increased from D1 to D2 to go down again from D2 to D3). This pattern suggests that beyond a certain amount of water and nutrients (due to irrigation with wastewater) the development of the willow root system is negatively affected, and although the aboveground biomass remained high in the D3 treatment, we believe that the development of the plant was somewhat unbalanced. No significant changes were found in the deep morphological traits related to irrigation with sewage wastewater.