Pyrethrum secondary metabolism: biosynthesis, localization and ecology of defence compounds

A.M. Ramirez

Research output: Thesisinternal PhD, WU


The use of botanical insecticides is in today’s world an attractive alternative to the less safe and environmentally malign synthetic chemicals, whose overall longer persistence in the environment does not only make them more contaminant, but also increases their chances of causing a rapid development of resistance in the target pest and having negative-side effects on beneficial organisms. Yet, at present only a handful of botanical products are in commercial use for insect control on crops. Among the most important botanical insecticides (pyrethrins, rotenone, neem and essential oils), pyrethrins have the longest history of effective use against a wide range of insects and best record of low toxicity to mammals. Although pyrethrins were relegated from their once prominent position in the mid-1930’s, the recent market trends towards “reduced risk” pesticides and host plant resistance have brought pyrethrins back to the attention and initiated the generation of knowledge around them. Pyrethrins refer to an oleoresin extracted from the dried daisy-like flowers of pyrethrum (Tanacetum cinerariifolium). The active constituents are six esters formed by a combination of two acids (chrysanthemic acid and pyrethric acid) and three alcohols (pyrethrolone, cinerolone and jasmolone, collectively called rethrolones).The esters of chrysanthemic acid with the rethrolones constitute type I pyrethrins, whereas the esters of pyrethric acid are collectively known as type II pyrethrins. Apart from being the source of pyrethrins, pyrethrum also produces a range of other defense compounds collectively known as sesquiterpene lactones, which have also been implicated in plant defense against herbivores, pathogens, and competing plant species. Although pyrethrins and sesquiterpene lactones are found throughout the whole plant, the highest concentrations of both types of compounds are found in the achenes of the flowers, which are densely covered with glandular trichomes. GC-MS analysis revealed that trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. Although glandular trichomes were known to participate in the production of mono- and sesquiterpene compounds that were stored in or emitted from the subcuticular cavity just outside the apical cells, here we demonstrate that basipetal secretion can also occur. In pyrethrum, the monoterpene-derived portion of pyrethrins, chrysanthemic acid (CA), is translocated from the trichomes to the pericarp, where it is esterified into pyrethrins that accumulate in the intercellular space. We also show that during seed maturation, pyrethrins stored in the pericarp are absorbed by the developing embryo, and that during seed germination these embryo-stored pyrethrins are recruited by the germinating seedling, which, due to the lack of trichomes, cannot produce defense compounds themselves. At early stages, not only sesquiterpene lactones that diffuse to the soil from the seed coat, but also the pyrethrins found in the seedlings, seem to play a more important role as antimicrobials than as insecticides.

Although there has been considerable progress on the chemistry of pyrethrins, the molecular/biochemical basis of their biosynthesis was largely unknown. The acid and alcohol moieties of pyrethrins derive from distinct pathways. Whereas the alcohol portion is believed to be derived from linolenic acid and share a large part of its biosynthetic pathway with jasmonic acid, the acid moieties are monoterpenes with a cyclopropane ring that are supposed to be derived from an irregular monoterpene pathway. Before the start  of this project, only one enzyme, chrysanthemyl diphosphate synthase (TcCDS), had been isolated and demonstrated to catalyze the first step in the biosynthesis of the acid portion of pyrethrins, which consist of the condensation of two molecules of DMAPP to produce chrysanthemol (COH) via chrysanthemyl diphosphate. During this project an additional acyltransferase enzyme (TcGLIP) was isolated by another group and demonstrated to be responsible for the esterification of (1R,3R)-chrysanthemoyl-CoA and (S)-pyrethrolone, one of the last steps in the biosynthesis of pyrethrins, which was demonstrated in this thesis to likely take place in the pericarp.

To identify additional genes of the pyrethrin biosynthetic pathway, we generated three EST libraries derived from ovaries, trichomes and leaves. Gene candidates were obtained either by keyword interrogation of the annotated contigs or by blasting the libraries with known genes catalyzing similar reactions in other plants. Given the likelihood of cytochrome P450s as potential candidates to catalyze the missing steps in the biosynthesis of the acid moiety of pyrethrins, the pyrethrum EST libraries, were first interrogated for genes encoding cytochrome P450 (CYP) enzymes with a developmental expression pattern similar to TcCDS, and a specific expression in CA-producing glandular trichomes. Experiments with yeast microsomes allowed the selection of two enzymes capable of converting COH into chrysanthemal. Although after agro-infiltration none of these enzymes affected the background level of CA, one of them (Ct21854) resulted in a strong reduction of COH emission, which correlates with a significantly higher amounts of a CA conjugate, confirming that Ct21854 qualifies as a chrysanthemic acid synthase efficiently converting COH into CA.

Rethrolones have been proposed to originate from linolenic acid and share part of the oxylipin pathway with jasmonic acid, which in turns implies that one of the first committed steps should involve a lipoxygenase enzyme, catalyzing the hydroperoxidation of linolenic acid at position 13 of the hydrocarbon chain. Based on this assumption the pyrethrum EST libraries were interrogated for genes encoding LOX enzymes. The expression patterns of twenty-five lipoxygenase EST contigs were characterized, and the ones with a developmental regulation similar to TcCDS and TcGLIP were selected. Subsequently, the molecular cloning of a lipoxygenase, TcLOX1, was carried out.  Recombinant TcLOX1was demonstrated to catalyze the peroxidation of the linolenic acid substrate at the C13  

position. The gene shares the developmental and trichome-specific  expression pattern with TcCDS, suggesting that a trichome production and translocation could be operating for the alcohol moiety of pyrethrins as well.

Finally, besides pyrethrins, pyrethrum plants are also a source of sesquiterpene lactones. Even though considerable information on bioactivity and industrial significance of pyrethrum sesquiterpene lactones is available, the localization and biosynthetic origins were largely unknown. Like in other species of the Asteraceae family, pyrethrum sesquiterpene lactones are exclusively stored in trichomes and it is shown that germacratrien-12-oic acid (GAA) is most likely the central precursor of all known sesquiterpene lactones found in pyrethrum. Candidate genes implicated in the first two committed steps leading from farnesyl diphosphate to GAA were retrieved from the pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. Furthermore, a gene encoding an enzyme capable of catalyzing the C6 hydroxylation of GAA was characterized. This hydroxylation results in spontaneous lactonization likely resulting in the putatively identified C6-C7-costunolide-derived STLs in pyrethrum. However, the enzyme may also catalyze the hydroxylation at the C6 position of the lactonized precursor of all reported C7-C8-type STLs.

In conclusion, the work compiled in this thesis presents new insights into the participation of pyrethrum trichomes in the biosynthesis and selective trafficking of pyrethrins precursors and STLs in opposite directions, and contributes to understanding the role of these flower-stored secondary metabolites in the immunization of the next generation against insect and fungal pathogens. Moreover, this study makes an important contribution towards understanding the biochemical and molecular bases of pyrethrins and STLs, the two most relevant bioactive compounds found in pyrethrum.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Bouwmeester, Harro, Promotor
  • Dicke, Marcel, Promotor
  • Jongsma, Maarten, Co-promotor
Award date3 May 2013
Place of Publication[S.l.]
Print ISBNs9789461735171
Publication statusPublished - 2013


  • tanacetum cinerariifolium
  • pyrethrins
  • metabolism
  • biosynthesis
  • defence mechanisms
  • plant physiology
  • defensive secretions
  • secretions


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