<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>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:13851840 |
| Date | 27 March 2019 |
| Creators | Barco, Brenden Lee |
| Publisher | Yale University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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