Browsing by Author "Hagel, Jillian M."

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    Functional genomics reveals novel o-demethylases involved in the biosynthesis of codeine and morphine in opium poppy
    (2009) Hagel, Jillian M.; Facchini, Peter J.
    Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The biosynthesis of morphine and related alkaloids in opium poppy occurs via a complex, multistep pathway beginning with the amino acid tyrosine. Corresponding genes encoding many of the enzymes involved in morphine biosynthesis have been isolated. However, molecular clones are not yet available for some enzymes, and enzyme activity accounting for two key O-demethylation steps leading from thebaine to morphine has yet to be detected. As part of a functional genomics platform aimed at isolating new genes, 1H nuclear magnetic resonance (NMR) metabolite profiling was used to characterize six varieties of opium poppy exhibiting altered alkaloid accumulation profiles. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principal component analysis of the 1H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety "P" and a high-thebaine, low-morphine cultivar "T." Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. This finding provided a rational basis for a microarray-based, comparative transcriptomics approach, wherein the transcriptome of T poppy stem was compared with those of high-morphine cultivars. This study led to the isolation of thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), which together represented the first identified 0-demethylases in the 2-oxoglutarate/Fe(II)-dependent dioxygenase family. It was shown that gene-specific silencing of T6ODM and CODM dramatically alters morphinan alkaloid profiles of opium poppy.
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    Metabolic engineering of hydroxycinnamic acid amide in nictoiana tabacum
    (2004) Hagel, Jillian M.; Facchini, Peter J.
    Feruloyltyramine and 4-coumaroyltyramine participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility or otherwise inhibit fungal growth. Hydroxycinnamoyl­CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) and tyrosine decarboxylase (TYDC) are purported to play key roles in the stress-induced regulation of tyramine­derived hydroxycinnamic acid amide (HCAAT) biosynthesis. Transgenic tobacco (Nicotiana tabacum cv Xanthi) was engineered to constitutively express tobacco THT. A T₁ plant over-expressing THT was crossbred with T₁ tobacco expressing opium poppy TYDC2 to produce a T₂ line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound feruloyltyrarnine and 4-coumaroyltyramine were examined. In roots, THT and TYDC activities were not substantially altered in transgenic lines beyond their relatively high wild type levels; thus, no changes in HCAAT precursor or product levels were detected. In leaves, increased TYDC activity resulted in a larger cellular pool of tyramine, and lower levels of phenylalanine. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The initial rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities. These results show that both THT and TYDC exert partial and synergistic control over the flux of intermediates involved in HCAAT biosynthesis under some conditions.