| In this dissertation a combination of mathematical modeling and experimental testing was used to study the regulation of monoterpene metabolism in peppermint. Due to the regulatory complexity and compartmentalization of plant metabolism, it is essential to understand which modeling approach is suitable to tackle a specific scientific problem. Thus, a comprehensive review which summarizes pros and cons of the various modeling approaches is included as the second chapter of this dissertation. For dynamic experimental systems, kinetic modeling is the most reliable strategy, since it accounts for the continuous diurnal, circadian and seasonal changes in the expression and activities of biosynthetic enzymes.High quality peppermint essential oils are characterized by a complex compositional balance of monoterpenes, with high quantities of (-)-menthol, moderate amounts of (-)-menthone and low levels of (+)-pulegone and (+)-menthofuran. However, under adverse environmental conditions such as low light intensity and drought, the branch point intermediate (+)-pulegone and the side product (+)-methofuran are accumulated, thus rendering an oil of inferior quality. Dynamic kinetic modeling of the peppermint monoterpene biosynthetic pathway suggested that competitive inhibition of pulegone reductase, the enzyme catalyzing the conversion (+)-pulegone into (-)-menthone, by (+)-menthofuran, could explain the monoterpene profiles obtained with plants grown under low light conditions. An experimental follow-up study using recombinant pulegone reductase indeed confirmed (+)-menthofuran as a competitive inhibitor of pulegone reductase.Peppermint plants expressing an antisense version of (+)-menthofuran synthase transcript were shown to contain low levels of (+)-pulegone and (+)-menthofuran. In addition to these desirable effects on essential oil composition, the oil yield in one particular line, designated MFS7, was also enhanced compared to wild-type controls. Based on real-time quantitative PCR assays, the monoterpenoid essential oil composition in MFS7 plants, but not increased yield compared to wild-type controls, could be explained by gene expression patterns. Interestingly, compared to controls, MFS7 plants had a higher density of glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. By integrating several levels of experimental data (glandular trichome distribution, biosynthetic gene expression patterns, and kinetic properties of biosynthetic enzymes) kinetic models accurately simulated experimentally determined monoterpene profiles. |
