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Tanacetum cinerariifolium flowers synthesize six pyrethrins that function as effective insecticides. trans-Chrysanthemol is an early intermediate in the synthesis of the monoterpene moiety of pyrethrins. Previously, the pyrethrum enzyme chrysanthemyl diphosphate synthase (TcCDS) was shown to catalyze the formation of the prenyl diphosphate compound chrysanthemyl diphosphate (CPP) by condensing two molecules of dimethylallyl diphosphate (DMAPP). Later work also showed that with a low concentration of DMAPP, TcCDS can also remove the diphosphate group to give chrysanthemol. The removal of the phosphate groups from other prenyl diphosphates, such as DMAPP, isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP), was previously shown to occur in two steps. In those cases, the first phosphate group is removed by a member of the Nudix hydrolase protein family, and the second by other unidentified phosphatases. These previously characterized Nudix proteins involved in the hydrolysis of prenyl diphosphates were shown to be cytosolic. Here we report that a plastidic Nudix protein from pyrethrum, designated TcNudix1, has high specificity for CPP and can hydrolyze it to chrysanthemol monophosphate (CMP). TcNudix1 is expressed specifically in the trichomes of the ovaries, where chrysanthemol is produced. TcNudix1 expression patterns and pathway reconstitution experiments presented here implicate the TcNudix1 protein in the biosynthesis of chrysanthemol.

Hundreds of orchid species secure pollination by sexually luring specific male insects as pollinators by chemical and morphological mimicry. Yet, the biochemical pathways involved in the synthesis of the insect sex pheromone-mimicking volatiles in these sexually deceptive plants remain poorly understood. Here, we explore the biochemical pathways linked to the chemical mimicry of female sex pheromones (chiloglottones) employed by the Australian sexually deceptive Chiloglottis orchids to lure their male pollinator. By strategically exploiting the transcriptomes of chiloglottone 1-producing Chiloglottis trapeziformis at distinct floral tissues and at key floral developmental stages, we identified two key transcriptional trends linked to the stage- and tissue-dependent distribution profiles of chiloglottone in the flower: (i) developmental upregulation of fatty acid biosynthesis and β-oxidation genes such as KETOACYL-ACP SYNTHASE, FATTY ACYL-ACP THIOESTERASE, and ACYL-COA OXIDASE during the transition from young to mature buds and flowers and (ii) the tissue-specific induction of fatty acid pathway genes in the callus (the insectiform odor-producing structure on the labellum of the flower) compared to the labellum remains (non-odor-producing) regardless of development stage of the flower. Enzyme inhibition experiments targeting KETOACYL-ACP SYNTHASE activity alone in three chiloglottone-producing species (C. trapeziformis, C. valida, and C. aff. valida) significantly inhibited chiloglottone biosynthesis up to 88.4% compared to the controls. These findings highlight the role of coordinated (developmental stage- and tissue-dependent) fatty acid gene expression and enzyme activities for chiloglottone production in Chiloglottis orchids.