No description available.

No description available.

No description available.

Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma. / Doctor of Philosophy / Plants produce a large number of chemicals that are important for growth, defense, flavor, and aroma. While chemical production has been studied in some major food crops (corn, tomato, rice), knowledge of the formation and function of chemicals in switchgrass and carrot is still limited. Switchgrass (Panicum virgatum L.), a grass of the Tallgrass Prairie, represents an important species grasslands of North America. Its natural resilience to stress has made switchgrass a preferred bioenergy crop. I found that switchgrass produces many compounds in the chemical class of terpenoids in roots and leaves that likely serve as a defense against damage from pests. In addition, I found that drought stress leads to the production of terpenoid compounds that may have roles in protection when water is limited. My research also demonstrates that roots of switchgrass and the related grass Setaria maintain substantial levels of the essential oil compound borneol. This terpenoid compound can act as a nutrient source for specific bacteria and/or an antimicrobial agent. Therefore, I proposed that switchgrass and Setaria roots produce borneol to establish a distinct root microbiome by recruitment of beneficial bacteria and deterrence of harmful microorganisms. To test this hypothesis, we genetically engineered plants to reduce borneol formation and accumulation in roots. Using these plants, we evaluated changes in the root microbiome in response to altered borneol levels. We found that interfering with borneol production in Setaria roots has limited influence on the microbiome inside roots. Although a similar approach was used for switchgrass, we were unable to significantly reduce borneol V formation in roots. Results from this study provide a better understanding of belowground plant-microbe interactions, and potential for enhancing resistance traits into other crop species. I also investigated the flavor and aroma compounds produced in carrots, which are considered a key supplemental vegetable due to high nutritional value and pleasant taste. Surprisingly, little has been known about the genetic factors that control flavor and aroma traits in colored carrot varieties. Therefore, I performed a robust characterization of the biosynthesis of terpenoids, which are the predominant aroma and flavor compounds in carrot. I identified several enzymes in carrot that can produce a diverse blend of terpenoids which are associated with sweet, spicy and bitter tastes. In addition, I discovered that carrot stems and leaves also maintain a rich chemistry of terpenoids similar to that in roots. Results from this work provide a baseline for engineering enhanced flavor in carrot and provide a deeper insight into essential oil formation in root crops.

Thesis (Ph. D.) -- Lincoln University, 2009. / Also available via the World Wide Web.

Thesis (Ph. D.)--Johns Hopkins University. / Includes bibliographical references.

Mean winter air temperatures have risen by 2.5˚C over the last 50 years in the northeastern U.S., reducing mean annual winter snowpack depth by 26 cm and the duration of winter snow cover by four days per decade. Because snow cover insulates soil from below-freezing air temperatures, continued declines in snowpack depth are projected to be accompanied by colder winter soil temperatures and more frequent soil freeze-thaw events. Soil bacteria and fungi will play a significant role in the forest ecosystem response to snowpack loss because they are the primary agents that carry out soil organic matter decomposition and soil nutrient cycling. Additionally, the effect of winter snowpack decline on soil bacterial and fungal communities may act indirectly via winter climate change effects on plant roots. The objectives of my dissertation research were to first determine the effect that reductions in winter snow cover has on microbial exoenzyme activity, microbial respiration, net nitrogen (N) mineralization, and net nitrification rates in two mixed-hardwood forests (Harvard Forest, MA and Hubbard Brook Experimental Forest, NH). Additionally, I sought to determine the relative role that abiotic factors (i.e., winter snow cover or soil frost) versus biotic factors (i.e., altered root-microbe interactions) contribute to overall changes in soil biogeochemical processes as winter snow cover declines. I found that winter snow depth and duration are related positively to microbial exoenzyme activity and microbial respiration following snowmelt in spring, but this relationship is transient and attenuates into the growing season. By contrast, soil freeze-thaw events during winter result in persistent declines in microbial oxidative enzyme activity that are not compensated for by warming soils during the growing season. Together, these results suggest that loss of winter snow cover will result in lower rates of nutrient cycling in northeastern U.S. hardwood forests. Tree roots interact with winter snow depth to affect net mineralization and nitrification rates, as well as bacterial and fungal community composition. Thus, winter climate change portends a reorganization of root-microbe interactions with important consequences for soil biogeochemical cycling in mixed hardwood forests of the northeastern U.S.

The relationship between a pathogen and its host is a complex series of events that occurs at the molecular level and is controlled by transcriptional and protein interactions. To facilitate the understanding of these mechanisms in Aspergillus flavus and Zea mays, three approaches were taken: 1) the development of a predicted interactome for Z. mays (PiZeaM), 2) the development of co-expression networks for Z. mays and A. flavus from RNA-seq data, and 3) the development of causal inference networks depicting interactions between the host and the pathogen. PiZeaM is the genome-wide roadmap of protein-protein interactions that occur within Z. mays. PiZeaM helps create a novel map of the interactions in Z. mays in response to biotic and abiotic stresses. To further support the predicted interactions, an analysis of microarray-based gene expression was used to produce a gene co-expression network. PiZeaM was able to capture conserved resistance pathways involved involved in the response to pathogens, abiotic stress and development. Gene Co-expression networks were developed by the simultaneous use of correlations to develop networks for differentially expressed genes, resistance marker genes, pathogenicity genes, and genes involved is secondary metabolism in Z. mays and A. flavus. From these networks, correlation and anti-correlation of host and pathogen gene expression was detected, revealing genes that potentially interact at different stages of pathogenesis. Finally, causal gene regulatory relationships were inferred using partial correlation analysis of Z. mays infected with A. flavus over a 3 day period. The gene regulatory network (GRN) sheds light on the specifics of the mechanisms of pathogenesis and resistance that govern the Z. mays-A. flavus interaction. The direct product of this research is the understanding of key transcription factors and signaling genes involved in resistance. This body of research highlights how PPIs and GRNs can be utilized to identify biomarkers and gene functions in both Z. mays and A. flavus.

No description available.

Crown gall disease of plants is caused by introduction of foreign DNA into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present in plant wound exudates. The exudates contain a number of phenolic compounds which act as chemical signals inducing expression of a number of genes directing the DNA transfer process. These are the virulence or vir genes, and vir::lac reporter gene fusions have been widely used to assay vir gene induction in Agrobacterium tumefaciens strains. Using such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or AS), and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, ("hydroxyacetosyringone" or HO-AS). However, in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression in A. tumefaciens was presented in a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate ring substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant wound exudates. Some Agrobacterium tumefaciens strains are restricted in host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component in grapevine wound exudates was purified. A newly described vir-inducing phenolic compound was isolated from a number of Vitis cultivars using gel filtration, thin layer and high pressure liquid chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester), using mass spectrometry. However, the presence of this compound in grapevine wound exudates does not provide a simple explanation for host range limitation of grapevine strains since it induces vir gene expression in both limited and wide host range strains of A. tumefaciens. Interestingly, neither AS nor HO-AS were present in grapevine-derived extracts. A convenient polyamide column chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene inducing mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS). GC-MS proved to be an ideal means for the identification of the phenolic components in partially purified extracts. Examination of wound exudates from a range of host and non-host species revealed that the production of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones in Nicotiana species. Wound exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induction of virE gene expression was examined in the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion plasmid pSM358cd was introduced into A. rhizogenes A4 by triparental mating and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes in A. rhizogenes. / Science, Faculty of / Botany, Department of / Graduate