D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

Reciprocal plantings of populations of broomsedge, taken from an abandoned strip mine and from an abandoned farm plot in south central Kentucky, on strip mine spoil and abandoned field soil resulted in patterns of populations differentiation. Clonal plantings of populations from strip mine habitats and old field development appeared equal in height and biomass when grown on old field soils in both field trials and growth chamber studies. Populations when planted in strip mine soils in field trials and controlled growth experiments. Later flowering in strip mine populations may be a key to survival strategy in the harsh microclimates of spoil banks. Andropogon virginicus L. may prove to be a significant and economically sound species in reclamation programs.

At least 110 million landmines have been planted since the 1970s in about 70 nations, many of which remain in place today. Some risk of detection may be mitigated using currently available remote sensing techniques when vegetation is present. My study focused on using plants as phytosensors to detect buried explosives. I exposed three species representing different functional types (Cyperus esculentus (sedge), Ulmus alata (tree), Vitis labrusca (vine)) to 500 mg kg-1 of Composition B (Comp B; 60/40 mixture of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT)), a commonly used explosive mixture, and measured functional traits and reflectance over a nine-week period. Cyperus esculentus was not a good indicator for the presence of explosive compounds. Comp B treatment woody species, U. alata and V. labrusca, exhibited changes in pigment content, leaf area, specific leaf area, dry leaf biomass, and canopy reflectance. The efficacy of plants as landmine detectors is species and/or functional group dependent.

In dioecious plants, selection due to sex function differences has produced sex-specific life histories, morphologies, and physiologies. In many dioecious seed plants, dimorphisms and population sex ratios have been plausibly linked, but similar links are not yet apparent in dioecious bryophytes. Population sex ratio bias is often expected to favor the sex with lower investment in sexual reproduction, especially in resource-poor environments. Unlike in seed plants, bryophyte males may have higher average reproductive investment than females, which typically have low offspring production rates due to sperm limitation. However, traits aside from reproductive investment such as shoot and leaf arrangement may be differentially selected and could influence life history and sex ratio, but these are rarely tested. My questions concentrated on the dimorphic traits responsible for sex ratio bias and their links to sex function. My studies, using the moss Bryum argenteum, included field and greenhouse experiments investigating sex ratio bias and morphological plasticity along a light/canopy openness (exposure) gradient, a greenhouse comparison of clump morphology and water-holding capacity, and a field and growth chamber study on sex-specific responses to stress (high temperature and desiccation). The sex ratio of urban Lexington, KY was highly female-biased, did not correlate with exposure, and was not linked with pre-zygotic reproductive investment. Leaf characteristics of B. argenteum plastically responded to exposure but were not sex-specific. However, juvenile females produced shoots at a faster rate and grew taller in high light. Juvenile male shoots held more external water than female shoots, but this did not predict mature clump water-holding capacity. Male clumps were shorter, denser, and held less water than females likely to shed sperm-laden water for sexual reproduction. Clump height did not trade off with reproductive investment, adding evidence that sex-specific size is linked with other aspects of sex function. Although chlorophyll fluorescence data (a measure of the status of photosystem II) from both field and growth chamber experiments indicated subtle sex-specific stress recovery responses among sexually immature and mature plants, differences were weaker than predicted and sexually mature shoots did not fare worse than vegetative shoots. The sex differences in size, clump morphology, and clump water-holding capacity very likely affect survival, growth, competitive ability, and ultimately adult sex ratio bias.

Doctor of Philosophy / Department of Biology / David C. Hartnett / Perennial grass populations propagate vegetatively via the belowground bud bank. Climate, photosynthetic pathway, and growth form impact bud production, longevity, and dormancy; leading to alterations in bud bank and tiller dynamics. Previous research in mesic C₄-dominated tallgrass prairie revealed that a C₄ grass had greater bud longevity and differing bud bank dynamics than a C₃ species. This study examined the bud bank dynamics of rhizomatous and caespitose grasses in a more arid C₃ dominated prairie to gain insights into how bud banks differ among grass species, growth forms, and environments, and the relationship between bud bank characteristics and grass architecture and growth patterns. The bud bank and tiller dynamics of four perennial grasses in the C₃-dominated northern mixed grass prairie were examined over 15 months. The C₃ caespitose and rhizomatous grasses produced similar numbers of buds per tiller and their bud longevity was [greater than or equal to] 2 years. Tiller longevity drove the turnover within the bud bank of the dominant C₃ caespitose grasses Hesperostipa comata and Nassella viridula. Their polycyclic tillers (tillers that lived for more than one year) created multi-aged bud banks. The rhizomatous C₃ grass Pascopyrum smithii also had a multi-aged bud bank because buds were able to live longer than its annual tillers. Differences between caespitose and rhizomatous C₃ grass bud banks were driven by differences in tiller and rhizome production and spatial distribution. Responses to water availability fluctuations are likely buffered by the maintenance of polycyclic tillers in the caespitose grasses and flexible timing of annual tiller recruitment in the rhizomatous grass. The C₄ rhizomatous grass Andropogon gerardii had similar phenology to populations in its tallgrass prairie range center. Despite declines in bud production per tiller and lowered flowering probability in mixed-grass prairie, A. gerardii maintained a multi-aged bud bank and a positive population growth rate via vegetative reproduction at both the center and edge of its range. Bud bank dynamics of different growth forms and photosynthetic pathways, as they offer insight into the control of grass population dynamics and production, will enhance understanding of the mechanisms by which management practices and environmental change can alter perennial grasslands.

Doctor of Philosophy / Department of Biology / Ruth Welti / The molecular mechanisms by which plants respond to environmental stresses to sustain growth and yield have great importance to agriculture. Lipid metabolites are a major element of plant stress responses. The model plant Arabidopsis thaliana is well-suited to study stress-driven compositional dynamics, metabolism, and functions of lipid metabolites. When Arabidopsis plants were subjected to wounding, infection by Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr), infection by Pseudomonas syringae pv. maculicola (Psm), and low temperature, and 86 oxidized and acylated lipids were analyzed using mass spectrometry, different sets of lipids were found to change in level in response to the various stresses. Analysis of plant species (wheat versus Arabidopsis), ecotypes (Arabidopsis Columbia 0 versus Arabidopsis C24), and stresses (wounding, bacterial infection, and freezing) showed that acylated monogalactosyldiacylglycerol was a major and diverse lipid class that differed in acyl composition among plant species when plants were subjected to different stresses. Mass spectrometry analysis provided evidence that oxophytodienoic acid, an oxidized fatty acid, is significantly more concentrated on the galactosyl ring of monogalactosyldiacylglycerol than on the glycerol backbone. A mass spectrometry method, measuring 272 lipid analytes with high precision in a relatively short time, was developed. Application of the method to plants subjected to wounding and freezing stress in large-scale experiments showed the method produces data suitable for lipid co-occurrence analysis, which identifies groups of lipid analytes produced by identical or inter-twined enzymatic pathways. The mass spectrometry method and lipid co-occurrence analysis were utilized to study the nature of lipid modifications and the roles of lipoxygenases and patatin-like acyl hydrolases in Arabidopsis during cold acclimation, freezing, and thawing.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Chad T. Miller / Kimberly A. Williams / Intumescences are a physiological disorder characterized by hypertrophy and possibly hyperplasia of plant cells. Many plant species are susceptible to intumescence development, but the specific causative factors remain uncertain. Ultimately, this disorder results in the death of the affected cells. Previous observations and research suggest that the quality and quantity of light to which plants are exposed may be a factor in development of the disorder. The purpose of the first study was to assess the preventive effect of UVB radiation on intumescence development in ornamental sweet potato (Ipomoea batatas). Two sweet potato cultivars, ‘Sidekick Black’ and ‘Ace of Spades,’ were grown under four light treatments of 1) Normal; 2) UVB; 3) UVB-Blocked; 4) Full-Spectrum. The ‘Ace of Spades’ cultivar was highly susceptible to intumescence development, while ‘Sidekick Black’ was much less susceptible to the disorder. For ‘Ace of Spades,’ the addition of UVB radiation significantly reduced the number of leaves affected with intumescences when compared to plants grown under the other light treatments. This study indicates a cultivar-specific effect of UVB light in minimizing intumescence development on ornamental sweet potato, therefore suggesting a potential genetic component in intumescence susceptibility. Many plant species are prone to similar physiological disorders in which lesions develop on the leaf tissue. Nomenclature for such lesions has varied significantly in the literature. Interchangeably using these terms causes confusion as to whether these names refer to the same or different disorders. The objective of the second study was to characterize the development of lesions on ornamental sweet potato (Ipomoea batatas ‘Blackie’), tomato (Solanum lycopersicum ‘Maxifort’) and interspecific geranium (Pelargonium בCaliente Coral’). Light microscopy, field emission scanning electron microscopy (FESEM), and digital photography were used to observe lesion development on each species. Lesions on ornamental sweet potato predominately involved the hypertrophy of the palisade parenchyma through the upper epidermis, while geranium lesions involved the hypertrophy of spongy parenchyma cells restricted by the lower epidermis. Tomato lesions involved both the hypertrophy and hyperplasia of the lower epidermis and spongy parenchyma. Thus, different species possess varied cellular responses when developing lesions due to physiological causes.

Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / The pathway for the movement of water through plants, from the soil matrix to the atmosphere, constitutes the hydraulic architecture of a plant. The linkage between the hydraulic architecture of woody plants and drought tolerance has received considerable attention, but much less work has been done on grasses. I investigated the linkage between the hydraulic architecture of grasses to physiological patterns of water use across a range of species and conditions. The rate of stomatal conductance (g[subscript]s) and photosynthesis (A) increased acropetally along the leaves of 5 grass species, which is a unique feature of this growth form. The internal structure of leaves also changed acropetally in order to minimize the pressure gradient across the mesophyll that would otherwise occur as a result of increasing g[subscript]s. The resistance to water movement through the mesophyll represented 80-90% of leaf resistance in six genotypes of Sorghum bicolor L. (Moench). This resistance was most important in controlling g[subscript]s and A when water was readily available, but as soil-moisture decreased it was the efficient transport of water through the xylem that was most important in maintaining plant function. I also investigated the relationship between hydraulic architecture and stomatal responses of grasses to increasing Vapor Pressure Deficit (D). Grasses with a larger proportion of their hydraulic resistance within the xylem were less sensitive to increasing D and plants with high root conductance maintained higher rates of gas exchange D increased. Finally, I investigated the tolerance of grasses to extreme drought events to test if there was a trade-off between drought tolerance and growth in grasses. Plants with drought tolerant leaf traits typically sacrificed the ability to move water efficiently through their leaves. Having drought tolerant leaves did not limit the plants ability to have high rates of gas exchange, and, in fact, the most drought tolerant plants had the high rates of g[subscript]s when expressed on a mass basis. Leaf-level drought tolerance did contribute to species’ occurrence, as the drought intolerant species I studied are not commonly found in low precipitation systems. The results presented here highlight the importance of studying the hydraulic architecture of plants to provide a better understanding of what controls plant function across a range of environmental conditions.

As pesquisas com os peptídeos hormonais de plantas se iniciaram na década de noventa com a descoberta da sistemina. Hoje existem diversos peptídeos identificados, e alguns deles já em avançado estágio de caracterização. O envolvimento desta classe de moléculas em diversas funções básicas e específicas da biologia dos vegetais despertou o interesse da comunidade científica. Dentre os peptídeos em fase de caracterização, destacam-se os representantes da família RALF. Os peptídeos RALF estão presentes em basicamente todo o reino vegetal, desde o musgo Physcomitrela pattens até as plantas superiores mono e dicotiledôneas. A conservação destes peptídeos no reino vegetal sugere um importante papel na fisiologia vegetal, e evidências recentes indicam a participação de RALF em processos básicos do desenvolvimento das plantas. O mecanismo pelo qual o peptídeo RALF atua e é percebido pela célula constitui etapa fundamental para sua caracterização funcional. Para tanto, no presente trabalho foram empregadas técnicas para identificação de proteínas de interação com RALF. Os resultados indicam que este peptídeo possivelmente tem sua atividade regulada pelo íon cálcio, através da interação com uma proteína de ligação a cálcio que, assim como o RALF, é secretada para o apoplasto. Estes dados colocam RALF em um cenário até o momento inédito no mecanismo de ação hormonal em plantas. A exemplo de animais e leveduras, observou-se também que o processamento de RALF ocorre em um sítio dibásico. A mutação de um dos aminoácidos deste sítio foi suficiente para impedir processamento do peptídeo in vivo e in vitro. A utilização de extratos protéicos da fração microsomal de Arabidopsis no ensaio in vitro indica que esta atividade é desempenhada por uma protease presente no sistema de endomembranas celular, provavelmente da classe das convertases. Os resultados publicados marcam o início dos estudos de caracterização do processamento de prohormônios em plantas. / The peptide hormone research has begun during the 90s decade with the systemin discovery. Nowadays several peptides have already been identified, and some of them are further characterized. The involvement of these molecules with a range of basic and specific biological functions has raised the scientific communitys interest. Among the peptides being studied, the RALF family is particularly intriguing. The RALF peptides can be found throughout the plant kingdom, from the moss Physcomitrella patens to the mono and dicot plant groups. The conserved occurrence of these peptides along the plant kingdom suggests an important role in the plant physiology field. Recent evidences indicate that RALF plays a role in basic mechanisms of plant development. The RALF mechanism of action and its perception by the cell are fundamental information in order to characterize this peptide function. In the present work experiments to identify RALF interacting proteins were employed. The results indicate that RALF peptides activity is possibly regulated by the calcium ion. This regulation is mediated by the interaction with a calcium binding protein. This calcium binding protein was found to be secreted to the apoplast. Presented data suggests that RALF is regulated by a mechanism never described before in the plant hormone research field. As previously described in animals and yeast the RALF propeptide processing takes place in a dibasic site. A single amino acid site specific mutation disrupted peptide processing in vivo and in vitro. The correct processing is mediated by proteases of the Arabidopsis microsomal fraction. This processing seems to occur at the endomembrane system, possibly catalized by a convertase class enzyme. The published results points the beginning of the peptide processing studies in plants.

SIP470, a putative tobacco lipid transfer protein, was identified in a yeast two-hybrid screen to interact with SABP2. SABP2 is a critical role in SA-mediated signaling in tobacco and other plants. In vitro studies using purified recombinant SIP470 confirmed that it is a lipid binding protein. In an attempt to determine its role in mediating stress responses, Arabidopsis T-DNA insertion knockout lines lacking SIP470 homolog were used for the analysis. These mutant plants were defective in basal resistance against microbial pathogens. Expression of defense gene PR-1 was also delayed in these mutant plants. Interestingly, these mutant plants were not defective in inducing systemic acquired resistance. Besides biotic stress, these mutant plants also showed increased susceptibility to abiotic stresses. To directly study the role of SIP470 in tobacco plants, transgenic tobacco lines, with reduced levels of SIP470 expression, were generated using RNAi and transgenic lines overexpressing SIP470 were also generated.