How Plants Synthesize Pyrethrins: Safe and Biodegradable Insecticides

Pyrethrin insecticides produced by Tanacetum cinerariifolium provide a human-safe and ecologically sound alternative to widely used synthetic insecticides.The development of transcriptomic and genomic resources for T. cinerariifolium facilitated the elucidation of numerous steps in the pyrethrin biosynthetic pathway.Pyrethrin biosynthetic genes have been engineered into diverse plant hosts; these efforts demonstrate the promise of engineering plants with improved endogenous defenses or of the development of an alternative source to pyrethrins for use as insecticides. Natural pyrethrin insecticides produced by Dalmatian pyrethrum (Tanacetum cinerariifolium) have low mammalian toxicity and short environmental persistence, providing an alternative to widely used synthetic agricultural insecticides that pose a threat to human health and the environment. A recent surge of interest in the use of pyrethrins as agricultural insecticides coincides with the discovery of several new genes in the pyrethrin biosynthetic pathway. Elucidation of this pathway facilitates efforts to breed improved pyrethrum varieties and to engineer plants with improved endogenous defenses or hosts for heterologous pyrethrin production. We describe the current state of knowledge related to global pyrethrum production, the pyrethrin biosynthetic pathway and its regulation, and recent efforts to engineer the pyrethrin pathway in diverse plant hosts. Natural pyrethrin insecticides produced by Dalmatian pyrethrum (Tanacetum cinerariifolium) have low mammalian toxicity and short environmental persistence, providing an alternative to widely used synthetic agricultural insecticides that pose a threat to human health and the environment. A recent surge of interest in the use of pyrethrins as agricultural insecticides coincides with the discovery of several new genes in the pyrethrin biosynthetic pathway. Elucidation of this pathway facilitates efforts to breed improved pyrethrum varieties and to engineer plants with improved endogenous defenses or hosts for heterologous pyrethrin production. We describe the current state of knowledge related to global pyrethrum production, the pyrethrin biosynthetic pathway and its regulation, and recent efforts to engineer the pyrethrin pathway in diverse plant hosts. Pyrethrins constitute a small class of specialized metabolites (see Glossary) produced in Dalmatian pyrethrum (Tanacetum cinerariifolium) and provide the plant with an effective endogenous chemical defense against insect herbivores and fungal pathogens [1.Grdisa M. et al.Chemical diversity of the natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia.Chem. 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Pyrethrins accumulate to 1–2% of dry mass in the mature flower heads, which are then harvested, dried, and powdered [1.Grdisa M. et al.Chemical diversity of the natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia.Chem. Biodivers. 2013; 10: 460-472Crossref PubMed Scopus (17) Google Scholar,55.Li J. et al.Comparative analysis of pyrethrin content improvement by mass selection, family selection and polycross in pyrethrum [Tanacetum cinerariifolium (Trevir.) Sch.Bip.] populations.Ind. Crop. Prod. 2014; 53: 268-273Crossref Scopus (13) Google Scholar]. The powdered material may then be marketed directly or extracted with organic solvents for formulation into insecticidal soaps and sprays [56.Ginsburg J.M. Kent C. The effect of soap sprays on plants.J. N. Y. Entomol. Soc. 1937; 45: 109-113Google Scholar]. 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A randomised, assessor blind, parallel group comparative efficacy trial of three products for the treatment of head lice in children – melaleuca oil and lavender oil, pyrethrins and piperonyl butoxide, and a "suffocation" product.BMC Dermatol. 2010; 10: 6Crossref PubMed Scopus (38) Google Scholar]. While T. cinerariifolium was originally harvested in its native Dalmatia in present-day Croatia, the crop was introduced into Japan in the late 19th century, and by the 1930s Japan produced most of the world's supply [3.Pares B. et al.Economic survey: the economic situation in Jugoslavia.Slav. Rev. 1925; 4: 491-505Google Scholar,61.Glassford J. The economics of pyrethrum.J. Econ. Entomol. 1930; 23: 874-877Crossref Google Scholar,62.Grunge W.H. Japan's pyrethrum position threatened.Far East. Surv. 1939; 8: 109-110Crossref Google Scholar]. More than a dozen countries have participated in industrial pyrethrum production, with significant sources of pyrethrum coming from Africa, Asia, Europe, and South America (http://www.fao.org/faostat/). However, Japan maintained a virtual monopoly on the pyrethrum market until World War II, after which East African nations took over most production. By the mid-1980s, Japanese pyrethrum production was negligible. Kenya dominated the pyrethrum market for the second half of the 20th century, but production dropped sharply in the mid-2000s. Currently, the major commercial pyrethrum producers are Rwanda, Tanzania, and the Australian state of Tasmania [63.Pethybridge S.J. et al.Diseases of pyrethrum in Tasmania: challenges and prospects for management.Plant Dis. 2008; 92: 1260-1272Crossref PubMed Scopus (50) Google Scholar,64.Ryan R.F. et al.Pyrethrum: the natural choice in pest control.in: Chung B. 1st International Symposium on Pyrethrum, the Natural Insecticide: Scientific and Industrial Developments in the Renewal of a Traditional Industry. International Society for Horticultural Science, 2015: 131-135Crossref Google Scholar]. Total levels of global industrial pyrethrum production have also fluctuated widely during the past half-century. Since the Food and Agriculture Organization of the United Nations (FAO) began keeping records of pyrethrum production in 1961, production has ranged from a record high of more than 30 000 metric tons in 1983 to an apparent low of less than 5000 tons in 2007, which marked the end of major production in Kenya. Production in Tanzania and Rwanda increased after this point, stabilizing global production. However, Australian production is not reported to the FAO and is thought to account for more than half of all global pyrethrum production [64.Ryan R.F. et al.Pyrethrum: the natural choice in pest control.in: Chung B. 1st International Symposium on Pyrethrum, the Natural Insecticide: Scientific and Industrial Developments in the Renewal of a Traditional Industry. International Society for Horticultural Science, 2015: 131-135Crossref Google Scholar], making reliable estimation of current global production difficult. FAO data indicates that global production excluding Australia totaled nearly 14 000 metric tons in 2017, the last year for which data are available. It is therefore likely that world production of pyrethrum approaches or exceeds the former 1983 record of 30 000 metric tons. Natural pyrethrins comprise six esters, each consisting of a monoterpenoid acid moiety conjugated to a rethrolone-type oxylipin alcohol (Figure 1A) [13.Laforge F. Barthel W. Constituents of pyrethrum flowers. 20. The partial synthesis of pyrethrins and cinerins and their relative toxicities.J. Org. Chem. 1947; 12: 199-202Crossref PubMed Scopus (4) Google Scholar,15.Godin P. et al.Insecticidal activity of jasmolin 2 and its isolation from pyrethrum (Chrysanthemum cinerariaefolium Vis).J. Econ. Entomol. 1965; 58: 548-551Crossref Google Scholar,65.Crombie L. Holloway S. Biosynthesis of the pyrethrins - unsaturated fatty-acids and the origins of the rethrolone segment.J. Chem. Soc. Perkin Trans. 1985; 1: 1393-1400Crossref Scopus (5) Google Scholar]. Their biosynthesis is known exclusively from T. cinerariifolium. Early feeding studies demonstrated that the pyrethrin biosynthetic pathway draws from two core plant metabolic pathways: the two monoterpenoids (chrysanthemic acid and pyrethric acid) are derived from the plastidial 1-deoxy-D-xylulose-5-phosphate (DXP) terpenoid pathway, while the three rethrolones (pyrethrolone, jasmolone, and cinerolone) are derived from the octadecanoid pathway (Figure 1B) [66.Matsuda K. et al.Biosynthesis of pyrethrin I in seedlings of Chrysanthemum cinerariaefolium.Phytochemistry. 2005; 66: 1529-1535Crossref PubMed Scopus (49) Google Scholar]. Pyrethrins containing chrysanthemic acid are termed 'type I' while those containing pyrethric acid are 'type II'. The three rethrolones termed pyrethrolone, jasmolone, and cinerolone are found in pyrethrin I and II, jasmolin I and II, and cinerin I and II, respectively. Pyrethrin I is the most abundant of the six pyrethrins in T. cinerariifolium while pyrethrin II is the second-most abundant (relative abundances of jasmolins and cinerins vary among T. cinerariifolium varieties) [1.Grdisa M. et al.Chemical diversity of the natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia.Chem. Biodivers. 2013; 10: 460-472Crossref PubMed Scopus (17) Google Scholar]. The full monoterpenoid pathway to chrysanthemic and pyrethric acids was recently elucidated with the aid of T. cinerariifolium transcriptomic and genomic resources (Figure 1B) [48.Xu H. et al.Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.New Phytol. 2019; 223: 751-765Crossref PubMed Scopus (18) Google Scholar,49.Xu H. et al.Coexpression analysis identifies two oxidoreductases involved in the biosynthesis of the monoterpene acid moiety of natural pyrethrin insecticides in Tanacetum cinerariifolium.Plant Physiol. 2018; 176: 524-537Crossref PubMed Scopus (28) Google Scholar,67.Yang T. et al.Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.J. Biol. 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Nearly two decades ago, it was shown that chrysanthemyl diphosphate (CDP) synthase (CDS) catalyzes the first step in the biosynthesis of these monoterpenoids via an unusual head-to-middle condensation of two dimethylallyl diphosphate (DMADP) units in plastids (Figure 1B) [4.Ramirez A.M. et al.Bidirectional secretions from glandular trichomes of pyrethrum enable immunization of seedlings.Plant Cell. 2012; 24: 4252-4265Crossref PubMed Scopus (47) Google Scholar,68.Rivera S.B. et al.Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4373-4378Crossref PubMed Scopus (82) Google Scholar]. Further conversion of CDP to the downstream acids requires dephosphorylation and oxidation; the action of one or more plastidial phosphatases was predicted to occur before the oxidation of chrysanthemol in the cytosol. However, while the initial characterization of CDS identified CDP as the reaction product, the results of subsequent investigations suggested that CDS might perform both the condensation of the two DMADP molecule precursors to give the cyclic monoterpene skeleton and the cleavage of the diphosphate group, yielding chrysanthemol [67.Yang T. et al.Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.J. Biol. Chem. 2014; 289: 36325-36335Crossref PubMed Scopus (30) Google Scholar]. This 'chrysanthemol synthase' model allowed diffusion of the first monoterpenoid product directly into the cytosol without invoking as-yet-undiscovered phosphatases. Recently, a Nudix-family phosphatase from T. cinerariifolium, Nudix1, was characterized that specifically dephosphorylates CDP yielding chrysanthemyl monophosphate (Figure 1B) [52.Li W. et al.A trichome-specific, plastid-localized Tanacetum cinerariifolium Nudix protein hydrolyzes the natural pyrethrin pesticide biosynthetic intermediate trans-chrysanthemyl diphosphate.Front. Plant Sci. 2020; 11: 482Crossref PubMed Scopus (6) Google Scholar]. Therefore, the involvement of this and other phosphatases, perhaps in addition to the dephosphorylating activity of CDS, cannot yet be ruled out [49.Xu H. et al.Coexpression analysis identifies two oxidoreductases involved in the biosynthesis of the monoterpene acid moiety of natural pyrethrin insecticides in Tanacetum cinerariifolium.Plant Physiol. 2018; 176: 524-537Crossref PubMed Scopus (28) Google Scholar,52.Li W. et al.A trichome-specific, plastid-localized Tanacetum cinerariifolium Nudix protein hydrolyzes the natural pyrethrin pesticide biosynthetic intermediate trans-chrysanthemyl diphosphate.Front. Plant Sci. 2020; 11: 482Crossref PubMed Scopus (6) Google Scholar]. Regardless of how chrysanthemol is generated from CDP, all evidence shows this compound as the branch point for the synthesis of both chrysanthemic acid and pyrethric acid. Once in the cytosol, the chrysanthemol hydroxyl can be directly modified by two oxidoreductases validated in vitro and in planta, alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 1 (ALDH1), which catalyze sequential oxidation of chrysanthemol to produce chrysanthemic acid (Figure 1B) [49.Xu H. et al.Coexpression analysis identifies two oxidoreductases involved in the biosynthesis of the monoterpene acid moiety of natural pyrethrin insecticides in Tanacetum cinerariifolium.Plant Physiol. 2018; 176: 524-537Crossref PubMed Scopus (28) Google Scholar]. A portion of the chrysanthemol pool can be hydroxylated at the C10 position by the cytochrome P450 chrysanthemol 10-hydroxylase (CHH) (CYP71BZ1), yielding the dihydroxylated compound 10-hydroxychrysanthemol [48.Xu H. et al.Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.New Phytol. 2019; 223: 751-765Crossref PubMed Scopus (18) Google Scholar]. The 10-hydroxyl group of this compound is converted to a carboxylic acid group by two additional oxidation steps catalyzed by CHH, yielding 10-carboxychrysanthemol, while the C1 hydroxyl group is oxidized to a carboxylic acid group by ADH2 and ALDH1, as in the biosynthesis of chrysanthemic acid described previously. Transient expression of the CDS, ADH2, ALDH1, and CHH genes in Nicotiana benthamiana leaves indicated that oxidation of the 10-hydroxy group to the carboxylic acid by CHH precedes oxidation of the C1 hydroxyl by ADH2 and ALDH1. The combined result of the actions of these three enzymes is 10-carboxychrysanthemic acid [48.Xu H. et al.Pyrethric acid of natural pyrethrin insecticide: complete pathway e