Identification, Characterization, and Functional Analysis of Terpenoid Specialized Metabolism in Switchgrass (Panicum virgatum) and Carrot (Daucus carota)
Muchlinski, Andrew Joseph
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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.
- Doctoral Dissertations