<p> Plants produce a variety of small molecules, including those essential for survival in all conditions (primary metabolites) or for more ecologically specific conditions (secondary metabolites). While primary metabolic pathways are broadly shared among plants, secondary metabolism is under constant selective pressure towards chemical innovation, given the continual fluctuation of the environment. Thus, plant secondary metabolism - whose constituents number in the hundreds of thousands - is lineage-specific, highly structurally diverse, and ultimately of high value to medicine, agriculture, and industry. Efforts to optimize the production of specific metabolites or to discover new compounds remain difficult primarily due to inadequate understandings of the metabolic genes involved and how these genes are regulated. This work first examines co-regulation, a major form of organization by which plant secondary metabolic genes are organized. In response to the bacterial crop pathogen <i>Pseudomonas syringae, Arabidopsis thaliana</i> and its relatives in the mustard family produce numerous secondary metabolites from the amino acid tryptophan, including the antimicrobial compound camalexin. However, hundreds of biosynthetic genes of unknown function are also simultaneously upregulated. Using metabolic profiling and co-expression analysis, I helped to identify the complete biosynthetic pathway to the indole-3-carbonylnitriles (ICNs), a previously unknown class of compounds. When the cytochrome P450 gene <i>CYP82C2</i> is mutated, biosynthesis of the compound 4-hydroxy-ICN (4OH-ICN) is abolished, and plant defense against <i>P. syringae</i> is impaired. Conversely, addition of 4OH-ICN to plants is sufficient to suppress bacterial growth. Next, this work examines the evolution of camalexin and 4OH-ICN metabolism. Cytochrome P450-directed secondary metabolism has been shown almost without exception to be evolutionarily derived from changes to enzymes with broad substrate specificity. By contrast, I observe through genetics, enzyme phylogenetic analysis, and transient expression assays that the ICN and camalexin biosynthetic pathways evolved from a common chemical substrate. In particular, changes to camalexin catalysis by the newly duplicated gene <i>CYP71A12</i> led to the formation of ICN metabolism in several mustard species, although both compounds are directly derived from indole cyanohydrin. Furthermore, 40H-ICN is an extremely recently evolved metabolite, derived from a flurry of genic, epigenetic and transposon-mediated rearrangements of a yet-more recent gene duplicate (<i>CYP82C2</i>). These regulatory changes to <i>CYP82C2</i> lead to its pathogen-inducibility solely in the species <i>A. thaliana</i>. I additionally identify WRKY33 and MYB51 as two sets of defense regulators that carefully fine-tune 40H-ICN metabolism by direct biosynthetic gene regulation. WRKY33 transcription factor, which is involved in the species-specific regulation of <i>CYP82C2</i>, is conserved throughout flowering plants, indicating that transcriptional recruitment is an important feature in the expansion of secondary metabolism. Finally, this work probes possible molecular functions of 40H-ICN and camalexin by exploring the molecular mechanisms underlying their secretion from roots and regulation of cell death processes. This study ultimately reveals that the proliferation of diverse chemical arsenals in plants is greatly aided by the regulatory capture of new and rapidly evolving genes by evolutionarily more stable transcription factors. Future emphases on transcriptional regulators of secondary metabolism may thus aid in the discovery of new secondary metabolic pathways on a more rapid scale.</p><p>

<p> Pierce&rsquo;s disease (PD), caused by bacterium Xylella fastidiosa, seriously hampers the cultivation of <i>Vitis vinifera</i> also known as bunch grapes, in different parts of the world. The bacterium clogs xylem vessels and forms a biofilm, resulting in the wilting of the plant. Bunch grape cultivars exhibit certain degree of tolerance to PD, however most commercial cultivars suffer heavy loss due to this devastating disease. Therefore, studies on genetic variation for disease tolerance will assist in identification of key molecular components that confer tolerance to PD. <i>Vitis</i> species, such as, Florida hybrid bunch (FH) and muscadine grape (<i> Vitis rotundifolia</i>) are widely cultivated in southeastern United States, and are known for their tolerance to PD. A detailed proteomic profile study of contrasting grape species is vital to understand the biological molecules associated with the PD tolerance. However information on total protein composition of <i>Vitis xylem</i> and sap is limited. The overall goals of this study are to determine the signal sequences associated with xylem and sap for the delivery of therapeutic proteins to control <i>Xylella fastidiosa. </i> The specific objectives of this research project are: 1) to compare the proteome profiles of xylem tissue and xylem sap from PD tolerant and -susceptible grapevine cultivars, and 2) to determine the role of proteins in the tissue and sap associated with PD tolerance mechanism. In this study, we used Bunch, FH, and Muscadine grape cultivars to characterize differentially expressed and unique proteins. Differentially expressed proteins were identified using LC MS/MS spectrometry searched against <i>Vitis</i> database. A total of 2519 and 402 proteins were identified in xylem and sap respectively, of which 151 proteins were common to both tissues. Bunch, FH, and muscadine sap showed 52, 53, and 30 unique proteins respectively. The cluster dendrogram analysis of the sap proteome showed that all of the <i>Vitis</i> species are bifolious. Based on the aforementioned, Florida hybrid bunch and muscadines are more closely related to each other than to bunch grape. Functional analysis and gene ontology revealed that proteins involved in carbohydrate metabolic process are more abundant in bunch grape, while FH and muscadine grape have more defense related proteins. Therefore, it is plausible to conclude that major functions of sap proteins in Bunch, FH, and Muscadine grapes are carbohydrate metabolic process and proteolysis (23%), protein phosphorylation (38%), and oxidation and reduction process (16%), respectively. Proteins involved in the defense and peroxidase activity are abundantly present in xylem and sap of FH and muscadine, and these proteins are relatively in reduced levels in bunch xylem and sap. Together, our findings highlight the possible roles of the identified unique proteins towards PD tolerance to Florida hybrid bunch and muscadine cultivars.</p>

Thesis (Ph. D.)--University of Toronto, 2008. / Includes bibliographical references.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / "Title from dissertation home page (viewed July 11, 2007)." Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4258. Adviser: Jeffrey D. Palmer.

This dissertation focuses on dioecious angiosperms with a genetic system of sex determination based on a pair of heteromorphic sex chromosomes. Silene latifolia and S. dioica (Caryophyllaceae), with their X/Y mechanism and heterogametic males, have become model organisms for the study of genetic sex determination in angiosperms. Random amplified polymorphic DNA (RAPD) markers provide a valuable tool for the study of the genetic nature of the male determining Y chromosome. We first embarked on a search for Y chromosome RAPD markers using the breeding technique of bulked segregant analysis, obtaining 101 Y chromosome markers which together, were shown to characterize the two species. Genetic mapping placed one of these Y chromosome molecular markers in the pseudoautosomal region of the sex chromosomes. This finding provides a tool for the study of recombination rates among sex chromosomes and relative rates of evolution of X and Y chromosomes. Finally, we investigated the molecular nature of a highly conserved Y chromosome marker and looked for homologous sequences in other related dioecious and hermaphroditic species. The marker of choice had been found to be conserved across males from four species of Silene, section Elisanthe. It was cloned, sequenced and analyzed by Southern hybridization. This Y chromosome marker is a high copy sequence that shares homology to female DNA. Forward and reverse primers were designed to amplify the male specific band only. The amplification pattern of the resulting sequence characterized amplified region (SCAR) in related taxa provide evidence for: (1) a common ancestry of the Y chromosomes of dioecious Silene, section Elisanthe, (2) a different branch of the evolution of dioecy in section Otites, (3) the distant relationship of the hermaphrodite S. noctiflora to the dioecious members of section Elisanthe and (4) homology between the Y chromosome of dioecious Silene dioica and autosomes of hermaphroditic S. flos-cuculi.

<p> This study analyzes the range-wide genetic diversity and population structure of American hart&rsquo;s-tongue fern (<i>Asplenium scolopendrium</i> var. <i>americanum</i>, AHTF), a rare fern species in the eastern United States. AHTF populations from New York, Michigan, Alabama, Tennessee, and Canada are examined using combined simple-sequence repeat and inter-simple sequence repeat markers. Genetic data provide insights on levels of genetic diversity, population structure, genetic differentiation, gene flow, total allele frequency, number of rare alleles, linkage disequilibrium and mating system. Overall, three main genetic clusters were identified, which are represented by: 1) populations from NY; 2) all three populations from Canada and the rest of the populations from the U.S.; and 3) the commercially available hart&rsquo;s-tongue fern. Genetic data is utilized to designate Evolutionary Conservation Units, Management Units and Relevant Genetic Units, particularly for the U.S. populations. This study recommends seven populations as priority for conservation and management in the U.S.</p>

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2706. Adviser: Geoffrey A. Levin. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

<p> Plant cell walls are under investigation as a source for biofuel production, yet conversion of cell walls (biomass) into biofuel is currently too expensive to be competitive with gasoline. Biomass is recalcitrant; that is, it resists enzymatic degradation by cellulases into monosaccharides such as glucose. One source of recalcitrance may be the presence of extensins, covalently bound cell wall proteins that are extremely insoluble.</p><p> To determine what influence, if any, extensins have on biomass recalcitrance, I performed several experiments. I first turned to poplar biomass, which is a model source for biofuels. I found that protease treatment of poplar biomass after liquid hot water pretreatment reduced the hydroxyproline content (a proxy for extensins). The reduction in hydroxyproline content correlated with reduced recalcitrance, seen as an increase in glucose release after cellulase digestion of poplar biomass. I also tested whether <i>Arabidopsis</i> T-DNA insertional mutations in the genes encoding enzymes that perform extensin post-translational modifications could reduce extensin content or cross-linking, and whether this reduction was associated with reduced biomass recalcitrance. I found that although these mutants were hypothesized to have reduced incorporation of extensin in cell walls, no significant effects on extensin content in inflorescence stem cell walls (an analog for woody biomass), nor on glucose release from biomass, were found in any mutant line. Finally, I looked at the effects of extensin overexpression on glucose release in transgenic Arabidopsis lines containing synthetic genes encoding the complete extensin domain from <i> SlLRX1</i> or a short C-terminal region of 20 amino acids of <i> SlLRX1,</i> fused to the red fluorescent reporter protein tdTomato. Observation of the tdTomato fluorescence in transgenic biomass after various chemical and enzymatic treatments indicated that the C-terminal 20 amino acids of <i> SlLRX1</i> are sufficient to allow a strong association with the cell wall, while the complete <i>SlLRX1</i> extensin domain leads to an even stronger, perhaps covalent linkage. Lines transformed with the complete <i> SlLRX1</i> extensin domain had more than twice the hydroxyproline content in their stems than wild-type, but this increase in hydroxyproline did not affect the amount of glucose released from stems upon cellulase digestion. </p><p> Since protease treatment reduced both hydroxyproline content and recalcitrance in poplar biomass, further experiments to assess the nature of the association between extensins and cell walls are warranted to attempt to further reduce recalcitrance. In the experiments I performed, the stems of extensin modification mutant Arabidopsis lines showed no change in extensin modification, and therefore no effect on recalcitrance was observed; stems of transgenic overexpression Arabidopsis lines showed increased extensin content, but again, no effect on recalcitrance was observed. My investigations in Arabidopsis focused on stem tissue, as this is analogous to material used in biofuel production. However, extensins are most abundantly expressed in roots in many plants, particularly in Arabidopsis. Examination of roots of both mutant and transgenic Arabidopsis may be more revealing of the interactions between extensins, cell walls, and recalcitrance.</p>

<p> The redistribution of nitrogen from old leaves to young leaves during senescence is crucial for plant survival. The <i>Arabidopsis thaliana </i> chloroplast residential protease S33Ab (At5g11650) was predicted to contribute to the degradation of chloroplast stromal proteins during senescence by bioinformatics tools. Rubisco degradation did not co-segregate with the T-DNA insertion mutant <i>s33Ab.</i> The location of T-DNA insertions into <i>S33Ac</i> (At1g18360) and <i>S33Ad</i> (At1g73480) were confirmed by sequencing. Total leaf proteins from <i>Ab, Ac, Ad </i> single, <i>Ab/Ac, Ab/Ad, Ac/Ad</i> double and <i> Ab/Ac/Ad</i> triple mutants were isolated from senescing leaves, subjected to immunoblot analysis to quantify Rubisco large subunit (LSU), glutamine synthase 2 (GS2), Rubisco activase (RCA), and light harvesting complex protein for photosystem II, b (Lhcb1) and compared to wild type. There is no significant difference among all the genotypes. This genetic analysis shows that S33Ab and its two closely related homologs, S33Ac and S33Ad do not individually contribute or work additively in chloroplast stromal protein degradation.</p>

<p> Auxin is a phytohormone that has long been known to control many aspects of plant growth and development. The YUCCA (YUC) gene family is a large group of genes that catalyze auxin biosynthesis and have been shown to be critical for vegetative growth and inflorescence development in grasses. There is genetic redundancy present with <i>Arabidopsis YUCs,</i> but in <i> Zea mays</i> (maize), a single gene knockout of <i>ZmSPI1</i> causes a severe inflorescence phenotype. Since <i>Oryza sativa</i> (rice), another grass species, does not show an inflorescence phenotype when <i> OsYUC1/SPI1</i> is knocked down, <i>SPI1</i> appears to have undergone an evolutionary shift in function within the grass family. This study shows that <i>SPI1</i> expression in PACMAD (Panicoideae, Arundinoideae, Chlorodoideae, Micrairoideae, Aristidoideae, and Danthoniodeae subfamilies) clade grasses <i>Sorghum bicolor</i> and <i>Setaria italica</i> occurs at sites of inflorescence branching and is consistent with maize, but in BEP (Bambusoideae, Ehrhartoideae, and Pooideae subfamilies) clade grasses rice and <i>Brachypodium distachyon SPI1</i> shifts from localized expression to more generalized expression and potentially becomes weaker. Artificial microRNA (amiRNA) knockdowns of <i>SPI1 </i> expression in <i>Brachypodium</i> did not show a phenotype when expression was reduced to 28.01% (+/- 6.39%) of wild type. In rice and <i> Brachypodium,</i> other <i>YUC</i> genes were shown to be expressed in the inflorescence by quantitative RT-PCR (qPCR), suggesting YUC proteins are more redundant in BEP grasses such as <i>B. distachyon</i> and <i> O. sativa,</i> than in maize and potentially its relatives.</p>