Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis. / Ph. D.

Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. / Ph. D.