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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/102778 |
| Date | 01 October 2019 |
| Creators | Muchlinski, Andrew Joseph |
| Contributors | Biological Sciences, Tholl, Dorothea, Haak, David C., Badgley, Brian D., Scharf, Birgit E., Zhao, Bingyu |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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