Date Available
12-14-2011
Year of Publication
2008
Document Type
Dissertation
College
Agriculture
Department
Plant Physiology
First Advisor
Joseph Chappell
Abstract
Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.
Recommended Citation
Lee, Sungbeom, "EXPLORING THE BIOCHEMICAL AND EVOLUTIONARY DIVERSITY OF TERPENE BIOSYNTHETIC ENZYMES IN PLANTS" (2008). University of Kentucky Doctoral Dissertations. 587.
https://uknowledge.uky.edu/gradschool_diss/587
