Pyrethrin Biosynthesis: The Cytochrome P450Oxidoreductase CYP82Q3 Converts JasmoloneTo Pyrethrolone1[OPEN]

Wei Li,a Daniel B. Lybrand,b Fei Zhou,a Robert L. Last,b,c and Eran Picherskya,2,3

aDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor,Michigan 48109bDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48823cDepartment of Plant Biology, Michigan State University, East Lansing, Michigan 48823

ORCID IDs: 0000-0002-8670-584X (W.L.); 0000-0003-3010-2203 (D.B.L.); 0000-0001-6974-9587 (R.L.L.); 0000-0002-4343-1535 (E.P.).

The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides known as pyrethrins. Pyrethrinsare esters consisting of an irregular monoterpenoid acid and an alcohol derived from jasmonic acid (JA). These alcohols,referred to as rethrolones, can be jasmolone, pyrethrolone, or cinerolone. We recently showed that jasmolone is synthesizedfrom jasmone, a degradation product of JA, in a single hydroxylation step catalyzed by jasmone hydroxylase (TcJMH). TcJMHbelongs to the CYP71 clade of the cytochrome P450 oxidoreductase family. Here, we used coexpression analysis, heterologousgene expression, and in vitro biochemical assays to identify the enzyme responsible for conversion of jasmolone to pyrethrolone.A further T. cinerariifolium cytochrome P450 family member, CYP82Q3 (designated Pyrethrolone Synthase; TcPYS), appeared tocatalyze the direct desaturation of the C1–C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS ishighly expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T. cinerariifolium genesinvolved in JA biosynthesis. Thus, as previously shown for biosynthesis of the monoterpenoid acid moiety of pyrethrins,rethrolones are synthesized in the trichomes. However, the final assembly of pyrethrins occurs in the developing achenes.Our data provide further insight into pyrethrin biosynthesis, which could ultimately be harnessed to produce this naturalpesticide in a heterologous system.

Pyrethrins are a group of six similar chemicals, synthe-sized by the plant pyrethrum to (Tanacetum cinerariifolium),which are very effective at immobilizing and killing flyinginsects (McLaughlin, 1973). These compounds fall intotwo groups. The first, called Type-I pyrethrins, includesjasmolin I, pyrethrin I, and cinerin I, and each containthe monoterpene acid transchrysanthemic acid, boundto one of three so-called rethrolones alcohols—respec-tively, jasmolone, pyrethrolone, and cinerolone (Fig. 1).Although there is some variability between different

cultivars, Type-I pyrethrins generally account for ap-proximately half or more of the total pyrethrins in theflower, with pyrethrin I being the most abundant Type-I pyrethrin (Head, 1973). The second class of pyrethrins,called Type II, are esters containing one of the samethree alcohols, but linked to pyrethric acid instead of tochrysanthemic acid, and are respectively called jasmo-lin II, pyrethrin II, and cinerin II (Fig. 1). Pyrethrins poseno danger to humans and most mammals. They arebiodegradable and photolabile, properties that slow theevolution of resistance in insects (Mitra et al., 1987). Theflowers of pyrethrum, where pyrethrin biosynthesis ismaximal, produce all six possible esters in varyingconcentrations (Head, 1973; Ramirez et al., 2012). Forthat reason, pyrethrum flowers serve as the source forcommercial extraction (Jones, 1973). However, costconsiderations limit the use of natural pyrethrins, andcheaper, synthetic pyrethrins, called pyrethroids, aremore heavily used, even though they are less biode-gradable and some pose more danger to mammalsand fish (Barthel, 1973; Sheets et al., 1994; DeMiccoet al., 2010).

The details of the enzymatic steps leading to the bi-osynthesis of the monoterpenoid acids chrysanthemicacid and pyrethric acid were recently elucidated (Riveraet al., 2001; Xu et al., 2018, 2019). Chrysanthemyl di-phosphate synthase (TcCDS) condenses two molecules

1This work was supported by the National Science Foundation(collaborative research grant no. 1565355 to E.P. and no. 1565232 toR.L.L.) and the National Institute of General Medical Sciences of theNational Institutes of Health (predoctoral training award grant no.T32-GM110523 to D.B.L.).

2Author for contact: lelx@umich.edu.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Eran Pichersky (lelx@umich.edu).

W.L., R.L.L., and E.P. designed the experiments; W.L., D.B.L., andF.Z. conducted the experiments; W.L., R.L.L., and E.P. wrote the ar-ticle; all authors edited the article.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00499

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of the universal terpene precursor dimethylallyl di-phosphate (DMAPP) to produce chysanthemyl diphos-phate. This product, considered an irregular terpenebecause of the “head to middle” condensation reac-tion of the two DMAPP molecules, is then converted tochrysanthemol by hydrolysis of the diphosphate group,

a reaction that can be catalyzed by TcCDS or by other,as yet unidentified, phosphatases (Rivera et al., 2001;Yang et al., 2014). Chrysanthemol is converted tochrysanthemic acid by the sequential activities of twodehydrogenases, alcohol dehydrogenase2 (TcADH2) andaldehyde dehydrogenase1 (TcALDH1; Xu et al., 2018).

Figure 1. The biosynthetic pathway ofpyrethrins as presently known, and pro-posed step(s) for the synthesis of pyrethro-lone in T. cinerariifolium. The synthesis ofJA from linolenic acid is catalyzed by li-poxygenase (TcLOX), allene oxidase syn-thase (TcAOS), allene oxide cyclase(TcAOC), cis (1)-12-oxo-phytodienenicacid reductase (TcOPR), and three roundsof b-oxidation. JA is metabolized to jas-mone in a set of as-yet unidentified reac-tions. Jasmone is converted to jasmolonein a reaction catalyzed by TcJMH (Li et al.,2018). Pyrethrolone differs from jasmo-lone by the presence of a double bondbetween v1-carbon and the penultimatecarbon of the side chain in the former. Theintroduction of this double bond in jas-molone could be accomplished by a singlereaction or by first hydroxylation and thendehydration. Chrysanthemic acid is gen-erated from two molecules of DMAPP insequential reactions catalyzed by TcCDS,TcADH, and TcALDH (Xu et al., 2018).Pyrethric acid is generated from two mol-ecules of DMAPP by the action of thesethree enzymes plus TcCHH and TcCCMT(Xu et al., 2019). The pyrethrin esters areformed in a reaction catalyzed by theGDSL lipase-like protein (TcGLIP), whichlinks the acid moiety to the alcohol moiety(Kikuta et al., 2012). Pyrethrins withchrysanthemic acid as the acid moiety arecalled “type-I” pyrethrins; pyrethrins withpyrethric acid as the acid moiety are called“type-II” pyrethrins.

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To obtain pyrethric acid from chrysanthemol, the ac-tivities of TcADH2 and TcALDH1, which form thecarboxylic group at C1, are combined with the ac-tivity of a cytochrome P450 oxidoreductase, namedchrysanthemol 10-hydroxylase (TcCHH), whichcatalyzes three consecutive oxidation reactions ofcarbon 10 to form 10-carboxychrysanthemic acid;10-carboxychrysanthemic acid is then converted topyrethric acid by the methylation of the 10-carboxylgroup by the SABATH-class methyltransferase named10-carboxychrysanthemic acid 10-methyltransferase(TcCCMT; Xu et al., 2019).

Some progress has also been made in elucidating thesynthesis of the alcohol moieties of pyrethrins. Labelingexperiments showed that they are derived from jas-monic acid (JA) via jasmone (Matsuda et al., 2005), andwe recently demonstrated that jasmolone is producedby hydroxylation of jasmone in a reaction catalyzed byjasmone hydroxylase (TcJMH; Li et al., 2018). Further-more, feeding pyrethrum flowers with jasmone led notonly to a large increase in the concentration of freejasmolone and jasmolin I, but also to a lesser increase inthe concentration of free pyrethrolone and pyrethrin Iand an even smaller but still statistically significantincrease in the concentration of cinerin I (Li et al., 2018).These results suggested that pyrethrolone as well ascinerolone are derived from jasmolone, although theroute was not determined.

The genes involved in the biosynthesis of chrys-anthemic acid, 10-carboxychrysanthemic acid, and jas-molone are active mostly in the trichomes of the ovaries(Ramirez et al., 2012; Li et al., 2018; Xu et al., 2018, 2019).In contrast, TcCCMT, responsible for the final step in

the synthesis of pyrethric acid, and TcGLIP, the geneencoding the GDSL lipase-like protein responsible forlinking the acidmoiety to the alcohol moiety to form thefinal pyrethrin molecules, are maximally expressed inthe pericarp of the ovaries (Kikuta et al., 2012; Xu et al.,2019). Pyrethrins are also constitutively made in theleaves at lower levels than in the flowers, but theirsynthesis in the leaves is induced by wounding ortreatment with methyl jasmonate (MeJA: Ueda andMatsuda, 2011).

Pyrethrolone differs from jasmolone solely by thepresence of a double bond between C1 and C2 in thepentyl side chain of the former (Fig. 1). This desatura-tion could come about by various mechanisms, in-cluding a direct extraction of two electrons or byhydroxylation of either C1 or C2 followed by dehy-dration (Fig. 1). One class of enzymes that is knownto carry out both desaturation and hydroxylation isthe cytochrome P450 oxidoreductase superfamily(Morikawa et al., 2006; Field and Osbourn, 2008;Mizutani and Ohta, 2010). To identify such enzymesinvolved in pyrethrin biosynthesis, we previously per-formed Pearson coexpression analysis on the RNA-sequencing and metabolic data bases from differentstages of pyrethrum flowers, leaves, and stems toidentify cytochrome P450 oxidoreductase genes whoseexpression pattern in pyrethrum flowers positivelycorrelated to some degreewith the expression of TcCDSand the synthesis of pyrethrins. A total of 12 genecandidates were selected whose coefficients werehigher than 0.75 using TcCDS as a reference (Li et al.,2018; Xu et al., 2018, 2019). We subsequently showedthat one of them, designated as CYP71BZ1, encoded

Figure 2. Assay for TcPYS activity ofthe candidate cytochrome P450 oxidore-ductases identified in our coexpressionanalysis. The assays were performed bytransiently expressing the candidate genesin N. benthamiana for 3 d via agro-bacterium infiltration and immersing theirleaves in 2 mM of jasmone for 24 h, af-ter which the leaves were ground andextracted with MTBE. Detection of jas-molone and pyrethrolone in the MTBEextracts were by GC-MS (total ion mode).The jasmolone and pyrethrolone stan-dards were obtained as described in Liet al. (2018). Pyrethrolone was detectedonly in leaves coexpressing candidategene DN144246 with TcJMH.

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TcCHH, the enzyme involved in pyrethric acid bio-synthesis (Xu et al., 2019). Another was shown to beCYP71AT148, or TcJMH, the gene encoding the enzymethat catalyzes the formation of jasmolone from jasmone(Li et al., 2018). TcJMH activity was demonstrated bytransiently expressing it in Nicotiana benthamiana leaveswhile feeding the leaveswith jasmone, and showing theproduction of jasmolone (Li et al., 2018). Subsequently,in vitro assays with microsomal preparations fromN. benthamiana plants expressing TcJMH directly dem-onstrated the conversion of jasmone to jasmolone(Li et al., 2018). Here, we extend the coexpressionanalysis of the cytochrome P450 oxidoreductase genesexpressed in pyrethrum flowers to identify the genepyrethrolone synthase (TcPYS), which encodes the cyto-chrome P450 oxidoreductase CYP82Q3, the enzymethat catalyzes the formation of pyrethrolone fromjasmolone.

RESULTS

Identification of TcPYS by Transient Expression ofCandidate Genes in N. benthamiana Leaves

We previously identified 12 cytochrome P450 oxi-doreductase genes by coexpression analysis as possiblyinvolved in pyrethrin biosynthesis (Li et al., 2018; Xuet al., 2018, 2019). We transiently expressed each oneof them separately in N. benthamiana leaves and fedjasmone to the leaves. Whereas the plants expressing

TcJMH produced jasmolone, no production of pyre-throlone was observed in any of the plant linesexpressing these 12 cytochrome P450 oxidoreductasecandidate genes, suggesting that pyrethrolone cannotbe directly produced from jasmone by a P450 enzyme.Furthermore, in previous experiments in which jas-mone was fed to pyrethrum stage-3 flowers, we ob-served that the levels of free jasmolone in the flowersincreased by 328%, whereas the concentration of freepyrethrolone increased by 49% (Li et al., 2018). Theseresults suggest that pyrethrolone may be a downstreamproduct of jasmolone.To test if pyrethrolone is produced from jasmolone

by the action of a cytochrome P450 oxidoreductase,we transiently coexpressed TcJMH with each of theremaining 10 functionally unassigned cytochrome P450oxidoreductase genes (i.e. in pairwise combinations) inN. benthamiana leaves immersed in a jasmone solution.After coexpression for 3 dwith the leaves immersed in ajasmone solution for the last 24 h of this period, theleaves were ground and extracted with methyl tert-butyl ether (MTBE) and the extract analyzed by gaschromatography-mass spectromery (GC-MS). Pyre-throlone was detected only in leaves in which TcJMHwas coexpressed with the cytochrome P450 oxidore-ductase gene initially annotated as DN144246 (Fig. 2).As expected, jasmolone production was observed in allgene combinations due to the activity of TcJMH (Fig. 2).GC-MS analysis of leaves expressing TcJMH withDN144246 did not reveal peaks indicating additionalnew products besides jasmolone and pyrethrolone

Figure 3. Generation of pyrethrin I in leaves ofN. benthamiana. Plants transiently coexpressed TcJMH, TcPYS, and TcGLIP for 3 dvia agrobacteria infiltration and were immersed in a solution containing 2 mM of jasmone and 200 mM of chrysanthemic acid,after which the leaves were ground and extracted with MTBE. Detection of jasmolone and pyrethrolone in theMTBE extracts wasby GC-MS (total ion mode). The jasmolin I and pyrethrin I standards were obtained as described in Li et al. (2018). Pyrethrin I wasdetected only in leaves expressing candidate gene DN144246, along with TcJMH and TcGLIP, and only when fed with bothchrysanthemic acid and jasmone.

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(Fig. 2; Supplemental Fig. S1). To identify possible in-termediates that might be glycosylated or simplyless volatile, we further analyzed the plant material intwo ways. First, we hydrolyzed the extracts withb-glucosidase and then analyzed the de-glycosylatedsamples by GC-MS. No additional peaks were detec-ted and there was no change in the concentrationamounts of jasmolone and pyrethrolone (SupplementalFig. S2). Second, we derivatized the hydrolyzed extractwith N-tert-butyldimethylsilyl-N-methyltrifluoroacetamideto protect all possible hydroxyl groups from dehydration.By checking the specific ion fragmentm/z5 194.0, onlysilylated jasmolone and pyrethrolone were detected. Incontrast, no peaks representing single or double sily-lation products of possible hydroxyl intermediateswere detected (Supplemental Fig. S3).

We then transiently coexpressed the three genesTcJMH, DN144246, and TcGLIP in N. benthamianaleaves fed with both jasmone and chrysanthemic acid.GC-MS analysis of extract of these leaves identified py-rethrin I in addition to jasmolin I (Fig. 3; SupplementalFig. S4). Based on these combined results, we designatedgene DN144246 as encoding TcPYS. TcPYS belongs tothe CYP82 family, and was given the official catalogdesignation CYP82Q3 by Dr. David Nelson (https://drnelson.uthsc.edu/CytochromeP450.html; Nelsonet al., 1996).

To further confirm TcPYS function, we stably coex-pressed TcPYSwith TcJMH in Arabidopsis (Arabidopsisthaliana), with expression of both genes independentlydriven by the 35S promoter. Resulting transgenic plants

were tested for the production of jasmolone and pyre-throlone (Fig. 4). Surprisingly, a small amount of jas-molone, but no pyrethrolone, was detected in extractsfrom leaves of control plants (i.e. Col-0 nontransgenicplants) fed with 50 mM of jasmone (Fig. 4). However,plants expressing TcJMH alone contained 7.5-foldgreater levels of jasmolone than control plants (and nopyrethrolone), whereas plants expressing both TcJMHand TcPYS contained pyrethrolone in addition to jas-molone (Fig. 4). These results are consistent with TcPYShaving a desaturase activity on jasmolone.

Biochemical Characterization of TcPYS

Microsomes of N. benthamiana leaves expressingTcPYS were prepared and tested for activity with pu-rified jasmolone prepared from hydrolyzed jasmolin Ias described in Li et al. (2018). After incubation over-night, the reaction solutions were extracted with MTBEand the extract analyzed by GC-MS. This analysisdetected pyrethrolone as the sole product (Fig. 5). Byincubating microsomes with different concentrations ofjasmolone for 2 h, the Km value for TcPYS with jasmo-lone was calculated to be 34.3 6 2.6 mM (means 6 SD,n 5 3; Supplemental Fig. S5). To test the substratespecificity of TcPYS, we selected several availablechemicals with similar structures, including jasmone,MeJA, 4-hydroxy-4-methyl-7-cis-decenoic acid g-lactone,cis-3-Hexenyl 3-methylbutanoate, cis-3-hexenyl benzoate,and dihydrojasmone. TcPYS microsomal preparations

Figure 4. Coexpression of TcJMH and TcPYS in Arabidopsis. A, GC-MS detection of jasmolone and pyrethrolone in a line stablyexpressing TcJMH (JMH), two lines expressing TcPYS (line PYS1 and PYS2), and two lines expressing both genes (JMH 3 PYS1and JMH3 PYS2).Wild-type Col-0 linewas used as control. TIC, total ion chromatogram; SIM, single ionmonitoring. B, RT-qPCRanalysis of relative TcJMH and TcPYS transcript levels in lines analyzed in (A). C, Quantification of jasmolone and pyrethrolonelevels in the respective lines; N.D., not detected. Data are presented as means 6 SD (n 5 3).

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did not catalyze the oxidation of any of these com-pounds, even after overnight incubations (Fig. 5).

Subcellular Localization of TcPYS

Because TcJMH was previously localized to the en-doplasmic reticulum (ER), we checked the subcellular

localization of TcPYS by fusing the open reading frameof TcPYS with GFP and transiently expressing thisconstruct in Arabidopsis protoplasts under the controlof the 35S promoter. Images obtained via confocal mi-croscopy showed the green signal to be associated withthe ER, as indicated by the overlap with the coex-pressed ER marker AtWAK2-mCherry (Zhou et al.,2017; Fig. 6).

Figure 5. In vitro activity and substrate specificity assays for TcPYS. In vitro conversion of jasmolone to pyrethrolone in mi-crosomes prepared fromN. benthamiana leaves transiently expressing TcPYS. Substrate specificity was tested on chemicals withsimilar structures including jasmone,MeJA, hydroxy-4-methyl-7-cis-decenoic acid g-lactone, cis-3-Hexenyl 3-methylbutanoate,cis-3-hexenyl benzoate, and dihydrojasmone. The reactionwas carried out overnight, after which the solutionwas extractedwithMTBE and the extract analyzed by GC-MS.

Figure 6. Representative results showing subcellular localization of TcPYS. All images are of the same cell. A, Detection of greenfluorescence from TcPYS-GFP fusion protein. B, Detection of red fluorescence from the ER marker AtWAK2-mCherry fusionprotein. C, Overlay of GFPand red fluorescence from (A) and (B). D, Detection of autofluorescence from chloroplasts. E, Bright-field microscopy of the cell under transmitted white light. Scale bars 5 10 mm.

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Tissue-Specific Expression of TcPYS

We previously showed that TcJMH converts jasmoneto jasmolone in the trichomes of the ovaries (Li et al.,2018). To determine the tissue-specific expression pat-tern of TcPYS, reverse transcription quantitative PCR(RT-qPCR) experiments were performed with tran-scripts obtained from five floral developmental stages(Xu et al., 2018) as well from leaf, stem, and root tissues.Disk and ray florets were separated in stage-4 and -5florets. The transcript analysis showed that TcPYS hada similar expression pattern to that of TcCDS in flowers,with the transcription levels peaking at stages 2 and 3,and transcript levels being barely detectable in leaf,stem, or roots (Fig. 7A).

To further identify inwhich parts of the flower TcPYSis expressed, we extracted RNA from stage-3 flowertrichomes, ovaries, and corollas from disc florets andthe entire ray florets for RT-qPCR analysis. RT-qPCRexperiments indicated that TcPYS transcripts arefound almost exclusively in the trichomes (Fig. 7B).

As our previous work showed that MeJA treatmentof leaves induces the transcription of genes involved inpyrethrin biosynthesis (Li et al., 2018), we tested TcPYSfor its inducibility by MeJA. RT-qPCR experiments onRNA collected from pyrethrum leaves treated withMeJA indeed showed a characteristic induction ofTcPYS byMeJA, with transcript increases peaking at 6 hto 12 h after MeJA application and decreases observedat 24 h (Fig. 7C).

Phylogenetic Tree of Cytochrome P450 CYP82 Family

Phylogenetic analysis indicated that TcPYS belongsto the CYP82 clade (Fig. 8), and thus it was named ac-cordingly as CYP82Q3. TcPYS is the first protein in theCYP82Q subfamily that has been biochemically char-acterized. TcPYS shares 61.7% sequence identity withits closest relative, CYP82Q1 from Stevia rebaudiana, aprotein that has not yet been functionally characterized.More distantly related, but functionally characterized,proteins in the CYP82 clade act on flavonoids, ter-penes, alkaloids, and coumarins (Siminszky et al.,2005; Kruse et al., 2008; Lee et al., 2010; Beaudoin

and Facchini, 2013; Winzer et al., 2015; Hori et al.,2018; Tian et al., 2018; Fig. 8).

DISCUSSION

Coexpression Analysis, Heterologous Expression, and InVitro Biochemical Assays Identify Pyrethrum CYP82Q3as TcPYS

Our previous work suggested that jasmolone isproduced in a one-step enzymatic reaction from jas-mone, and that pyrethrolone is probably producedfrom jasmone in a multistep pathway, likely includingjasmolone as an intermediate (Li et al., 2018). Here, weemployed the N. benthamiana heterologous expressionsystem to coexpress TcJMH, the CYP protein that con-verts jasmone to jasmolone, together with candidateCYP genes identified by coexpression analysis. This ledto identification of TcPYS, which encodes an enzymethat converts jasmolone to pyrethrolone.N. benthamianaleaves coexpressing TcJMH and TcPYS were shown toproduce pyrethrolone upon feeding with jasmone.TcPYS is CYP82Q3, a member of the plant-specificCYP82 clade, which includes proteins known to cata-lyze the modification of other terpenes as well as fla-vonoids, coumarins, and alkaloids—all plant-specificspecialized compounds. Additional evidence for theenzymatic activity of TcPYS in converting jasmolone topyrethrolone was obtained by coexpression of TcJMHand TcPYS in transgenic Arabidopsis as well as byin vitro assays ofN. benthamianamicrosomes expressingTcPYSwith jasmolone as a substrate. The in vitro assaysof TcPYS with jasmolone and related compoundsalso indicated that the ER-localized enzyme is highlyspecific for jasmolone. Finally, N. benthamiana leavescoexpressing TcJMH, TcPYS, and TcGLIP producedpyrethrin I upon being fed both jasmone andchrysanthemic acid.

TcPYS May Act as a Desaturase

The oxidative conversion of jasmolone to pyrethro-lone could proceed via a hydroxyl intermediate followed

Figure 7. TcPYS transcription abundancein different tissues and in leaf under MeJAtreatment. A, RT-qPCR analysis of TcPYStranscripts in different developmentalstages of the flower (“T” and “B” representray florets and disk florets, respectively),leaf, stem, and root. B, RT-qPCR analysisof TcPYS transcripts in different parts ofstage-3 flowers. C, RT-qPCR analysis of2-week–old leaves treated with MeJA.Data are presented as means6 SD (n5 3or 4).

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by dehydration or directly by the removal of two elec-trons and two protons (Fig. 1). The various analyses ofthe products of the in vitro reaction (Figs. 3–5;Supplemental Figs. S2 and S3) identified only pyre-throlone. Whereas it is not possible to rule out pro-duction of short-lived, hard-to-detect hydroxylatedintermediates and an endogenous N. benthamiana en-zyme that acts on such an intermediate, these resultssuggest that TcPYS catalyzes the direct formation of aC–C double bond between the terminal carbons of thejasmolone pentyl side chain. If so, it would be the first

characterized member of the CYP82 subfamily thatacts as a desaturase. Most of these proteins catalyzehydroxylation reactions,with some exceptions:CYP82N2/N4 catalyzes a cyclization (Beaudoin and Facchini, 2013;Fujiwara and Ito, 2017), CYP82Y2 catalyzes a regiospecificisomerization (Winzer et al., 2015), CYP82G1 catalyzesoxidative degradation, and CYP82E4v1 catalyzes de-methylation (Siminszky et al., 2005; Lee et al., 2010).Direct desaturation catalyzed by CYP P450 enzymes

is uncommon. In most reactions catalyzed by theseenzymes, the activated enzyme-bound oxygen-iron

Figure 8. AMaximum Likelihood phylogenetic tree of TcPYS (CYP82Q3) and the proteins most closely related to it from CYP82family with functional assignments. The tree also includes TcCHH and TcJMH, which are involved in pyrethrin biosynthesis, andCYP94B3, which works on jasmonoyl-L-Ile and CYP710A1 as representative P450 enzymes with desaturase activities. Sourcesand functions are as follows: CYP82D62, flavone-6-hydroxylase (Ocimum basilicum); CYP82D33, flavone-6-hydroxylase(O. basilicum); CYP82D2, 49-deoxyflavones hydroxylase (Scutellaria baicalensis); CYP82D1.1, 49-deoxyflavones hydroxylase(S. baicalensis); CYP82D113, 7-keto-d-cadinene 18-hydroxylase (Gossypium raimondii); CYP82Q3, TcPYS (T. cinerariifolium);CYP82Q1 (S. rebaudiana); CYP82P3, dihydrosanguinarine 10-hydroxylase (Eschscholzia californica sspp. californica);CYP82P2, dihydrosanguinarine 10-hydroxylase (E. californica sspp. californica); CYP82C4, fraxetin 5-hydroxylase (Arabidopsis);CYP82C2, fraxetin 5-hydroxylase (Arabidopsis); CYP82N2, protopine 6-monooxygenase (E. californica); CYP82Y2,1,2-dehydroreticulinium reductase (Papaver somniferum); CYP82N5 N-methystylopine hydroxylase (E. californica sspp. californica);CYP82N4, methyltetrahydroprotoberberine 14-monooxygenase (P. somniferum); CYP82G1, (3E)-4,8-dimethyl-1,3,7-nonatrienesynthase (Arabidopsis); CYP82E4v1, nicotine demethylase (Nicotiana tabacum); CYP71BZ1, chrysanthemol 10-hydroxylase(T. cinerariifolium); CYP71AT148, TcJMH (T. cinerariifolium); CYP94B3, jasmonoyl-L-Ile hydroxylase (Arabidopsis); CYP710A1,C22-sterol desaturase (Arabidopsis). The tree is drawn to scale (per the number of substitutions per site).

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complex ([FeO]31) attacks a carbon (or N or S) atomand abstracts a hydrogen from it, forming a paired[FeOH]31–carbon radical. This is followed by the newlyformed hydroxyl rebounding to the carbon, generatinga C-OH group. In some rare cases, the abstraction of ahydrogen might be followed by the abstraction of asecond hydrogen from an adjacent carbon atom, fol-lowed by the formation of a double bond betweentwo atoms, along with the release of a water mole-cule (Guengerich, 2001). One such example is de-saturation at C22 of sterols in fungi and plants,which is catalyzed by CYP61 and CYP710 enzymes,respectively (Kelly et al., 1997; Morikawa et al.,2006). However, C22 of sterols, while on a sidechain, is not a terminal or penultimate carbon. Thedirect desaturation of valproic acid catalyzed by ratCYP3A1 is a more similar reaction to the formationof the double bond between the v1-carbon and thev2-carbon on the side chain of jasmolone by TcPYS(Fisher et al., 1998). Note that in valproic acid oxi-dation, both hydroxylation and desaturation hap-pen when the initial attack by the enzyme occurs atv2-carbon, but when the initial attack occurs on thev1-carbon, the result is 100% hydroxylation (Fisheret al., 1998).

Pyrethrin Biosynthesis Is a Multiorganellar andMulticellular Process

Maximal production of pyrethrins occurs in the py-rethrum flower, andmore specifically in the developingachenes (McLaughlin, 1973). The ovaries contain trichomes,where chrysanthemic acid, 10-carboxychrysanthemic acid,and at least the alcohol jasmolone are produced (Riveraet al., 2001; Li et al., 2018; Xu et al., 2019). The finalmethylation reaction in the synthesis of pyrethric acidas well as the ester formation of the pyrethrins occursmostly in the pericarp of the ovaries, and the assembledpyrethrins accumulate throughout the achenes and theseeds, evidently for protection (Ramirez et al., 2012;Xu et al., 2019). Even in the trichomes, the synthesis ofthese components is divided into multiple organelles.Chrysanthemol is produced in the plastid, whereas theoxidation steps leading to the monoterpene acids occurin the cytosol (Yang et al., 2014; Xu et al., 2018). JA, theprecursor of the rethrolones (i.e. jasmolone, pyrethro-lone, and cinerolone), is produced from linolenic acidpartly in the plastid, partly in the ER, and partly in theperoxisome, whereas the production of jasmolone fromjasmone occurs on the ER (Song et al., 1993; Schalleret al., 2000; Ziegler et al., 2000; Matsuda et al., 2005;Ramirez et al., 2013; Li et al., 2018). Here we show thatTcPYS is also predominantly expressed in pyrethrumflowers of developmental stages 2 and 3 as found forother pyrethrin biosynthetic genes such as TcCDS andTcJMH (Fig. 7A). Furthermore, TcPYS exhibits a patternof cellular expression (high levels of transcripts in tri-chomes) and subcellular protein localization (ER locali-zation) that is very similar to that of the TcJMH gene and

its protein, which generates jasmolone, the substrate ofTcPYS (Figs. 6 and 7).

Pyrethrins are also synthesized in pyrethrumleaves, although at a low level compared to that inflowers. However, the biosynthesis of pyrethrins inleaves is induced by wounding as well as by thewounding hormone jasmonate (Ueda and Matsuda,2011; Kikuta et al., 2012). Here we showed that TcPYSexpression is also jasmonate-inducible (Fig. 7C),similar to the expression of other pyrethrin biosyn-thetic genes (Li et al., 2018).

Overall, the data presented here suggest that TcPYSmay be an unusual cytochrome P450 oxidoreductasecapable of directly introducing a double bond in theside chain of jasmolone to produce pyrethrolone. Withthe recent discovery of TcJMH, the enzyme involved inthe synthesis of jasmolone from jasmone, and the set ofenzymes involved in the conversion of chrysanthemolto chrysanthemic acid and pyrethric acid, only thesynthesis of cinerolone remains unsolved in the bio-synthetic pathway of pyrethrins.

MATERIALS AND METHODS

Plant Growth Condition

Pyrethrum (Tanacetum cinerariifolium) plants were grown on soil in growthchambers under the following conditions: 25°C, 16 h, 400 mmol m22 s21 lightand 20°C, 8-h dark.Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana)plants were grown on soil in growth chambers at 22°C, 16 h, 150 mmol m22 s21

light and 20°C, 8-h dark in a growth room.

Transient Expression in N. benthamiana andJasmone Feeding

Cytochrome P450 oxidoreductase gene candidates were inserted into vectorpEAQ-HT and mobilized into Agrobacterium tumefaciens strain GV3101 as de-scribed in Li et al. (2018). Agrobacterium cells were cultured in LB media over-night until the OD value reached 1.0, then collected by centrifugation andresuspended in equal volume of buffer containing 10 mM of MES at pH 6.8,10 mM of MgCl2, and 100 mM of Acetosyringone (MMA buffer). Agrobacteriumcells were injected into 4-week–old N. benthamiana leaves via the abaxial sur-face. When performing coexpression of several genes, equal volumes of sus-pensions of differentAgrobacterium cells weremixed together for injection. After3 d, the leaves were harvested for feeding experiment or preparation ofmicrosomes.

In chemical feeding, two N. benthamiana leaves were immersed in 20 mL of2-mM jasmone, placed under vacuum at 12.5 psi for 10 min, and incubated for24 h at room temperature. After incubation, leaves were rinsed twice withwater, blotted dry with filter paper, and the leaves put into a 15-mL centrifugetube. MTBE (0.5 mL) was added to the tube and the leaves homogenized with amicro homogenizer to break the tissues. The homogenate was left at roomtemperature for 1 h, and the samplewas centrifuged at 12,000g for 5min and thesupernatant moved to a 2-mL bottle with a 0.25-mL glass insert for GC-MSanalysis.

For thehydrolysis andderivatizationexperiments, 36 leaveswerefirst soakedin an aqueous solution containing 2mM of jasmone for 24 h. Afterward, the leaveswere homogenized and 40 mL of extract buffer (acetonitrile/isopropanol/water:3:3:2) was added and the solution and incubated overnight at 4°C. The solutionwas next centrifuged at 20,000g for 10 min, and supernatant was lyophilized andresuspended in 1 mL of a solution of 0.1-M sodium acetate at pH 5.0. Four mg($8 units, one unit liberates 1mmol of Glc from salicin permin at pH 5.0 at 37°C)of almond b-glucosidase (cat. no. G0395; Sigma-Aldrich), which is known toefficiently hydrolyze b-D-glycoside from a wide range of substrates, was addedto 250-mL extract, and incubated for 4 h at 37°C. The lysate was next extrac-ted with 250 mL of MTBE for GC-MS analysis. For derivatization, 50 mL of

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hydrolyzed extract was dried in a speed vacuum device, and 100 mL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was added to the dry material,which was then incubated at 80°C for 4 h, and analyzed directly on GC-MS.

Arabidopsis Stable Expression

The sequences of TcJMH and TcPYS were inserted into the vector pSAT4A,which contains a CaMV 35S promoter and a CaMV 35S terminator, by doubledigest with EcoRI and BamHI and ligated with T4 DNA ligase. Next, the com-plete constructs (promoter–gene–terminator) were obtained by cutting withI-SceI and integrating the fragment into the binary vector pPZP-RSCII. Thebinary vectors were moved into Agrobacterium GV3101 for Arabidopsis floraldip transformation. TcJMH and TcPYS were transferred into Arabidopsis in-dependently, and the two overexpressing lines were crossed to obtain thecoexpression lines. Arabidopsis gene AtActin8 was used as internal referencegene to confirm transcription level of TcJMH and TcPYS in transgenic plants.Primers used are listed in Supplemental Table S1.

GC-MS Analysis and Km Value Measurement

GC-MS analysis was performed on GCMS-QP5000 (Shimadzu) with aTR-5MS column. The column temperature was programmed as follows: 50°Chold for 2 mins, 50°C to 330°C at 10°C min21, then hold 10 min. See our pre-vious work for pyrethrolone, jasmolone, jasmolin I, and pyrethrin I purification(Li et al., 2018).

Purification of microsomes was performed using methods published inSchaller (2017). Kinetic parameter measurements were obtained from 50-mLreactions containing 100 mM of Tris at pH 7.5, 300 mM of NADPH, 40 mL ofmicrosome, and varying concentration of jasmolone (0/25/50/100/200 mM) or100 mM of substrate for specificity test. After incubation for 2 h (for Km valuedetermination) or overnight (to determine substrate specificity) at room tem-perature, products were extracted with 100 mL of MTBE for GC-MS analysisand their concentrations plotted to verify enzyme saturation. The Km value wascalculated by hyperbolic regression analysis method with software Hyper32(https://hyper32.software.informer.com/). Data are presented as means 6 SD

(n 5 3).

RT-qPCR of Floral Transcripts and MeJA Induction ofTranscripts in Leaves

RNA was extracted with an E.Z.N.A. Plant RNA kit (cat. no. R6827-02;Omega Bio-tek). Reverse transcription reaction was carried out with a high-capacity cDNA reverse transcription kit (cat. no. 4368814; Thermo Fisher Sci-entific). RT-qPCRwas performedwith Power SYBRGreen PCRmaster mix (cat.no. 436759; Thermo Fisher Scientific). Different stages of Pyrethrum flowerwere assessed as described in Xu et al. (2018). Isolation of floral trichomes wasperformed using published methods from Ramirez et al. (2012) and MeJA in-duction was done as we reported in Li et al. (2018). Transcriptional level ofTcPYSwas determined by comparisonwith internal reference genes TcGAPDH,TcActin7, and TcTubulin3. Primers used are listed in Supplemental Table S1.

Subcellular Localization

Full-length TcPYS gene sequence was integrated into the expression vectorpEZS-NL using the primers listed in Supplemental Table S1. Arabidopsisprotoplasts preparation, transformation, and confocal microscopy were per-formed as described in Zhou et al. (2017).

Phylogenetic Analysis

Aminoacid sequences of functionally elucidatedCYP82 familyproteinsweredownloaded from the National Center for Biotechnology Information. AMaximumLikelihoodphylogenetic treewas produced by the software programMEGA7, which is based on the Jones–Taylor–Thornton matrix model (Kumaret al., 2016).

Accession Numbers

The sequence of TcPYS has been submitted to the National Center for Bio-technology Information under accession no. MG874675.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Comparisons of mass spectra of pyrethrolonegenerated from different sources.

Supplemental Figure S2. Analysis of possible glycosylation of rethrolonesin N. benthamiana leaves expressing TcJMH and TcPYS.

Supplemental Figure S3. GC-MS analysis of derivatized rethrolones inN. benthamiana leaves expressing TcJMH and TcPYS.

Supplemental Figure S4. Comparisons of mass spectra of pyrethrin I fromdifferent sources.

Supplemental Figure S5. An in vitro enzyme saturation curve for the con-version of jasmolone to pyrethrolone by purified microsome prepara-tions from N. benthamiana leaves expressing TcPYS.

Supplemental Table S1. Primers used in this investigation.

ACKNOWLEDGMENTS

We thankDr. DavidNelson fromUniversity of Tennessee for naming TcPYSas CYP82Q3. We also thank Dr. A. Daniel Jones and Dr. Anthony Schilmillerfrom Michigan State University for derivatization suggestions.

Received July 20, 2019; accepted August 16, 2019; published August 26, 2019.

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