Figure 1.
Simplified scheme for the formation of germacrane, eudesmane and guaiane-type sesquiterpene lactones.
The germacrane skeleton is formed from farnesyl diphosphate (FDP) and undergoes a cyclization reaction to either the guaiane or the eudesmane skeleton [43].
Figure 2.
Proposed biosynthetic routes to C6–C7 and C7–C8 sesquiterpene lactones.
I, Cyclization of farnesyl diphosphate (FPP) to (+)− germacrene A (GA) by (+)− germacrene A synthase (TcGAS). II, Oxidation of GA via germacra-1(10),4,11(13)-trien-12-ol (GOH) and germacra-1(10),4,11(13)-trien-12-al (GAL) into germacra-1(10),4,11(13)-trien-12-oic acid (GAA) catalyzed by an NADPH-dependent single P450 enzyme (TcGAO). III, Hydroxylation at the C6 position of GAA or of various C7–C8 lactones by a second P450 enzyme (TcCOS). IV, Hydroxylation at the C8 position of GAA, and V, subsequent spontaneous lactonization. VI, VII, and VII involve extra oxidative steps and esterification with glycosyl, tigloyl and acyl groups, and cyclization of the sesquiterpene backbone to get to the structures in Figure 3 and 4.
Figure 3.
Major sesquiterpene lactones reported for pyrethrum.
A, β-cyclopyrethrosin (1), chrysanin (2), pyrethrosin (3), dehydro-β-cyclopyrethrosin (4), chrysanolide (5) [3]. B, (11R)-11,13-dehydro-tatridin-A (6), (11-R)-6-O-β-D-glucosyl-11–13 dehydro-tatridin-B (7), (11R)-11,13-dehydro-tatridin-B (8) tatridin-A (9), and tatridin-B (10) [4].
Figure 4.
Glandular trichome content of pyrethrum achenes.
A, GC-MS analysis of chloroform dips of seed extracts, representing the content of trichomes. B, Putative chemical structures of the STLs found in pyrethrum trichomes according to a NIST library search. 1.8-hydroxy-3, 8a-dimethyl-5-methylene-2-oxododecahydronaphthol(2,3-b)furan-4yl acetate (p3), 1.8-hydroxy-8a-methyl-3,5-dimethylene-2-oxododecahydronaphthol(2,3-b)furan-4yl(2E)-2-methyl-2-butenoate (p7), and 2-methylbut-2enoic acid (5,8-dihydroxy-5,8a-dimethyl-3-methylene-2-oxo-dodecahydronaphtho(2,3-b)furan-4-yl) ester (p6/p8). The prefix “p” stands for putative. Compound p2 is probably an oxygenated sesquiterpene, p4 and p5 are likely to be STLs of undetermined structure, and p1 is an unknown compound. Table S1 provides further details. C, cryo-scanning electron microscopy image of complete, unopened disk florets showing the highest density of glandular trichomes in the indentations between the ribs of the ovaries (left panel) and closer view of trichomes (right panel). Ov, ovary; Co, corolla; Tr, trichome.
Figure 5.
Pyrethrum sesquiterpene lactones content in different tissues.
Peak areas of a C7–C8 type sesquiterpene lactone (Grey bars: compound p7 of Figure 4), and of a STL of undetermined structure Black bars: compound p5 of Figure 4) in pyrethrum leaves, stems, ray florets and disk florets relative to the peak areas in disk florets (100%) (A); and in pyrethrum flowers in different developmental stages relative to the peak areas in stage 5 flowers (B). Images of the corresponding pyrethrum flower heads (C) Error bars represent SEM (N = 3).
Figure 6.
Phylogenetic analyses of the germacrene A synthases and oxidases.
A, tree based on the deduced amino acid sequences of the putative pyrethrum germacrene A synthase (TcGAS) and other characterized plant GAS genes. B, tree based on the putative pyrethrum germacrene A oxidase (TcGAO) and costunolide synthase (TcCOS) and other characterized plant GAO and COS genes. The tree was constructed by Neighbour-Joining method using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). The species abbreviations are Ci, Cichorium intybus; Ha, Helianthus annuus; Ls, Lactuca sativa; Tp, Tanacetum parthenium; Tc, Tanacetum cinerariifolium.
Figure 7.
Germacrene A production in planta.
A, headspace analysis of volatiles emitted from Nicotiana benthamiana leaves agro-infiltrated with TcGAS. B, mass fragmentation pattern of compound 1 (Ba) and β-elemene from the Wiley library (Bb). C, cope rearrangement of germacrene A to β-elemene by heat.
Figure 8.
α-costic acid and γ-costic acid production in yeast.
A, GC-MS chromatograms for the terpenoids with a parent mass of m/s 234 isolated from yeast transformed with the indicated genes. Front line showing the metabolites for the empty vector control, middle line showing the metabolites of yeast transformed with TcGAS and TcGAO, and last line showing the metabolites of yeast transformed with Tanacetum parthenium TpGAS and Cichorium intybus CiGAO. B, mass fragmentation pattern of peak 1 (1a), α-costic acid (1b), peak 2 (2a), and γ-costic acid (2b). C, acid induced rearrangement of germacrene A acid to α-costic acid and γ-costic acid.
Figure 9.
Costunolide production in planta.
LC-QTOF-MS analysis of non-volatiles metabolites from Nicotiana benthamiana leaves agro-infiltrated with empty vector, TcGAS, TcGAS+TcGAO, and TcGAS+TcGAO+TcCOS. The MS spectrum of the two new peaks (peak 22.21 and peak 22.66) in leaves agro-infiltrated with pyrethrum GAS+GAO+COS and their respective parent ions ([m/z]− 352.1613 and 538.2184, respectively) are shown.
Figure 10.
Log2 of the Relative Gene Expression of pyrethrum TcGAS, TcGAO and TcCOS in different tissues.
A, Expression in ovaries isolated from flowers at different developmental stages (O2 to O7). B, expression in seedlings, leaves and trichomes isolated from stage 3 ovaries. Black bars represent TcGAS, grey bars represent TcGAO and white bars represent TcCOS. The Ct value for each sample was normalized using GAPDH as housekeeping gene. Error bars represent SEM (N = 3).