1

Short title: pyrethrolone formation catalyzed by a P450 enzyme 1

2

Pyrethrin biosynthesis: The cytochrome P450 oxidoreducatse CYP82Q3 converts 3

jasmolone to pyrethrolone1 4

5

6

7

Wei Lia, Daniel B. Lybrandb, Fei Zhoua, Robert L. Lastb, c, Eran Pichersky2, a 8

9 aDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann 10

Arbor, MI, USA. 11

12 bDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing 13

MI, USA 14 15 cDepartment of Plant Biology, Michigan State University, East Lansing MI, USA 16

17

ORCID IDs: 0000-0002-8670-584X (W.L.); 0000-0003-3010-2203 (D.L.); 0000-0003-2992-18

0380 (F.Z.); 0000-0001-6974-9587 (R.L.L.); 0000-0002-4343-1535 (E.P.) 19

20 1This work was supported by the National Science Foundation collaborative research grants 21

1565355 to EP and 1565232 to RLL as well as by a predoctoral training award to DBL from 22

Grant Number T32-GM110523 from the National Institute of General Medical Sciences of the 23

National Institutes of Health. 24

25 26 2Address correspondence to: Eran Pichersky, email: lelx@umich.edu 27

28

One-sentence summary: Cytochrome P450 CYP82Q3 from Tanacetum cinerariifolium is 29

involved in the introduction of the double bond in the side chain of jasmolone to generate 30

pyrethrolone, a component of pyrethrin insecticide. 31

Plant Physiology Preview. Published on August 26, 2019, as DOI:10.1104/pp.19.00499

Copyright 2019 by the American Society of Plant Biologists

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32

Key words: natural pesticides; plant biochemistry; specialized metabolism; cytochrome P450 33

34

Contact information for all authors: 35

Wei Li : weilium@umich.edu 36

Daniel Lybrand: lybrandd@msu.edu 37

Fei Zhou: feizhou@umich.edu 38

Robert Last: lastr@msu.edu 39

Eran Pichersky: lelx@umich.edu 40

41

The author responsible for distribution of materials integral to the findings presented in this 42

article in accordance with the policy described in the Instructions for Authors 43

(www.plantphysiol.org) is: Eran Pichersky (lelx@umich.edu). 44

45

Author contributions 46

W.L., R.L.L and E.P. designed the experiments; W.L., D.L., and F.Z. conducted the experiments; 47

W.L., R.L.L. and E.P wrote the article; all authors edited the article. 48

49

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Abstract 50

The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides 51

known as pyrethrins. Pyrethrins are esters consisting of an irregular monoterpenoid acid and an 52

alcohol derived from jasmonic acid. These alcohols, referred to as rethrolones, can be jasmolone, 53

pyrethrolone, or cinerolone. We recently showed that jasmolone is synthesized from jasmone, a 54

degradation product of jasmonic acid, in a single hydroxylation step catalyzed by jasmone 55

hydroxylase (TcJMH). TcJMH belongs to the CYP71 clade of the cytochrome P450 56

oxidoreductase family. Here, we used coexpression analysis, heterologous gene expression, and 57

in vitro biochemical assays to identify the enzyme responsible for conversion of jasmolone to 58

pyrethrolone. A further T. cinerariifolium cytochrome P450 family member, CYP82Q3 59

(designated Pyrethrolone Synthase; PYS), appeared to catalyze the direct desaturation of the C1-60

C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS is highly 61

expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T. 62

cinerariifolium genes involved in jasmonic acid biosynthesis. Thus, as previously shown for 63

biosynthesis of the monoterpenoid acid moiety of pyrethrins, rethrolones are synthesized in the 64

trichomes. However, the final assembly of pyrethrins occurs in the developing achenes. Our data 65

provide further insight into pyrethrin biosynthesis, which could ultimately be harnessed to 66

produce this natural pesticide in a heterologous system. 67

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68

Introduction 69

70

Pyrethrins are a group of six similar chemicals, synthesized by the plant pyrethrum (Tanacetum 71

cinerariifolium), which are very effective at immobilizing and killing flying insects (McLaughlin, 72

1973). These compounds fall into two groups. The first, called Type I pyrethrins, includes 73

jasmolin I, pyrethrin I, and cinerin I, and each contain the monoterpene acid trans-chrysanthemic 74

acid, bound to one of three so-called rethrolones alcohols – respectively jasmolone, pyrethrolone, 75

and cinerolone (Figure 1). Although there is some variability between different cultivars, Type I 76

pyrethrins generally account for approximately half or more of the total pyrethrins in the flower, 77

with pyrethrin I being the most abundant Type I pyrethrin (Casida, 1973). The second class of 78

pyrethrins, called Type II, are esters containing one of the same three alcohols, but linked to 79

pyrethric acid instead of to chrysanthemic acid, and are respectively called jasmolin II, pyrethrin 80

II, and cinerin II (Figure 1). Pyrethrins pose no danger to humans and most mammals. They are 81

biodegradable and photolabile, properties that slow the evolution of resistance in insects (Mitra 82

et al., 1987). The flowers of pyrethrum, where pyrethrin biosynthesis is maximal, produce all six 83

possible esters in varying concentrations (Head, 1973; Ramirez et al., 2012). For that reason, 84

pyrethrum flowers serve as the source for commercial extraction (Jones, 1973). However, cost 85

considerations limit the use of natural pyrethrins, and cheaper, synthetic pyrethrins, called 86

pyrethroids, are more heavily used, even though they are less biodegradable and some pose more 87

danger to mammals and fish (Barthel, 1973; Sheets et al., 1994; DeMicco et al., 2010). 88

89

The details of the enzymatic steps leading to the biosynthesis of the monoterpenoid acids 90

chrysanthemic acid and pyrethric acid were recently elucidated (Rivera et al., 2001; Xu et al., 91

2018; Xu et al., 2019). Chrysanthemyl diphosphate synthase (TcCDS) condenses two molecules 92

of the universal terpene precursor dimethylallyl diphosphate (DMAPP) to produce chysanthemyl 93

diphosphate. This product, considered an irregular terpene because of the “head to middle” 94

condensation reaction of the two DMAPP molecules, is then converted to chrysanthemol by 95

hydrolysis of the diphosphate group, a reaction that can be catalyzed by TcCDS or by other, as 96

yet unidentified, phosphatases (Rivera et al., 2001; Yang et al., 2014). Chrysanthemol is 97

converted to chrysanthemic acid by the sequential activities of two dehydrogenases, alcohol 98

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dehydrogenase 2 (TcADH2) and aldehyde dehydrogenase 1 (TcALDH1) (Xu et al., 2018). To 99

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obtain pyrethric acid from chrysanthemol, the activities of TcADH2 and TcALDH1, which form 100

the carboxylic group at C1, are combined with the activity of a cytochrome P450 oxidoreductase, 101

named chrysanthemol 10-hydroxylase (TcCHH), which catalyzes three consecutive oxidation 102

reactions of carbon 10 to form 10-carboxychrysanthemic acid. 10-Carboxychrysanthemic acid is 103

then converted to pyrethric acid by the methylation of the 10-carboxyl group by the SABATH-104

class methyltransferase named 10-carboxychrysanthemic acid 10-methyltransferase (TcCCMT) 105

(Xu et al., 2019). 106

107

Some progress has also been made in elucidating the synthesis of the alcohol moieties of 108

pyrethrins. Labeling experiments showed that they are derived from jasmonic acid (JA) via 109

jasmone (Matsuda et al., 2005), and we recently demonstrated that jasmolone is produced by 110

hydroxylation of jasmone in a reaction catalyzed by jasmone hydroxylase (TcJMH) (Li et al., 111

2018). Furthermore, feeding pyrethrum flowers with jasmone led not only to a large increase in 112

the concentration of free jasmolone and jasmolin I, but also to a lesser increase in the 113

concentration of free pyrethrolone and pyrethrin I and an even smaller but still statistically 114

significant increase in the concentration of cinerin I (Li et al., 2018). These results suggested that 115

pyrethrolone as well as cinerolone are derived from jasmolone, although the route was not 116

determined. 117

118

The genes involved in the biosynthesis of chrysanthemic acid, 10-carboxychrysanthemic acid 119

and jasmolone are active mostly in the trichomes of the ovaries (Ramirez et al., 2012; Li et al., 120

2018; Xu et al., 2018; Xu et al., 2019). In contrast, TcCCMT, responsible for the final step in the 121

synthesis of pyrethric acid, as well as TcGLIP, the gene encoding the GDSL lipase-like protein 122

responsible for linking the acid moiety to the alcohol moiety to form the final pyrethrin 123

molecules, are maximally expressed in the pericarp of the ovaries (Kikuta et al., 2012; Xu et al., 124

2019). Pyrethrins are also constitutively made in the leaves at lower levels than in the flowers, 125

but their synthesis in the leaves is induced by wounding or treatment with MeJA (Ueda and 126

Matsuda, 2011). 127

128

Pyrethrolone differs from jasmolone solely by the presence of a double bond between C1 and C2 129

in the pentyl side chain of the former (Figure 1). This desaturation could come about by various 130

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mechanisms, including a direct extraction of two electrons or by hydroxylation of either C1 or 131

C2 followed by dehydration (Figure 1). One class of enzymes that is known to carry out both 132

desaturation and hydroxylation is the cytochrome P450 oxidoreductase superfamily (Morikawa 133

et al., 2006; Field and Osbourn, 2008; Mizutani and Ohta, 2010). To identify such enzymes 134

involved in pyrethrin biosynthesis, we previously performed Pearson coexpression analysis on 135

the RNA-seq and metabolic data bases from different stages of pyrethrum flowers, leaves, and 136

stems to identify cytochrome P450 oxidoreductase genes whose expression pattern in pyrethrum 137

flowers positively correlated to some degree with the expression of TcCDS and the synthesis of 138

pyrethrins. A total of 12 gene candidates were selected whose coefficients were higher than 0.75 139

using TcCDS as a reference (Li et al., 2018; Xu et al., 2018; Xu et al., 2019). We subsequently 140

showed that one of them, designated as CYP71BZ1, encoded TcCHH, the enzyme involved in 141

pyrethric acid biosynthesis (Xu et al., 2019). Another was shown to be CYP71AT148, or TcJMH, 142

the gene encoding the enzyme that catalyzes the formation of jasmolone from jasmone (Li et al., 143

2018). TcJMH activity was demonstrated by transiently expressing it in Nicotiana benthamiana 144

leaves while feeding the leaves with jasmone, and showing the production of jasmolone (Li et al., 145

2018). Subsequently, in vitro assays with microsomal preparations from N. benthamiana plants 146

expressing TcJMH directly demonstrated the conversion of jasmone to jasmolone (Li et al., 147

2018). Here, we extend the coexpression analysis of the cytochrome P450 oxidoreductase genes 148

expressed in pyrethrum flowers to identify the gene pyrethrolone synthase (TcPYS), which 149

encodes the cytochrome P450 oxidoreductase CYP82Q3, the enzyme that catalyzes the 150

formation of pyrethrolone from jasmolone. 151

152

153

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154

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Results 155

Identification of Pyrethrolone Synthase by Transient Expression of Candidate Genes in 156

Nicotiana benthamiana Leaves 157

158

We previously identified 12 cytochrome P450 oxidoreductase genes by co-expression analysis as 159

possibly involved in pyrethrin biosynthesis (Li et al., 2018; Xu et al., 2018; Xu et al., 2019). We 160

transiently expressed each one of them separately in N. benthamiana leaves and fed jasmone to 161

the leaves. Whereas the plants expressing TcJMH produced jasmolone, no production of 162

pyrethrolone was observed in any of the plant lines expressing these 12 cytochrome P450 163

oxidoreductase candidate genes, suggesting that pyrethrolone cannot be directly produced from 164

jasmone by a P450 enzyme. Furthermore, in previous experiments in which jasmone was fed to 165

pyrethrum stage 3 flowers, we observed that the levels of free jasmolone in the flowers increased 166

by 328% whereas the concentration of free pyrethrolone increased by 49% (Li et al., 2018). 167

These results suggest that pyrethrolone may be a downstream product of jasmolone. 168

169

To test if pyrethrolone is produced from jasmolone by the action of a cytochrome P450 170

oxidoreductase, we transiently coexpressed TcJMH with each of the remaining 10 functionally 171

unassigned cytochrome P450 oxidoreductase genes (i.e., in pairwise combinations) in N. 172

benthamiana leaves immersed in a jasmone solution. After coexpression for 3 days with the 173

leaves immersed in a jasmone solution for the last 24 h of this period, the leaves were ground 174

and extracted with MTBE and the extract analyzed by GC-MS. Pyrethrolone was detected only 175

in leaves in which TcJMH was coexpressed with the cytochrome P450 oxidoreductase gene 176

initially annotated as DN144246 (Figure 2). As expected, jasmolone production was observed in 177

all gene combinations due to the activity of TcJMH (Figure 2). GC-MS analysis of leaves 178

expressing TcJMH with DN144246 did not reveal peaks indicating additional new products 179

besides jasmolone and pyrethrolone (Figure 2, Supplemental Figure S1). To identify possible 180

intermediates that might be glycosylated or simply less volatile, we further analyzed the plant 181

material in two ways. First, we hydrolyzed the extracts with β-glucosidase and then analyzed the 182

de-glycosylated samples by GC-MS. No additional peaks were detected and there was no change 183

in the concentrations amounts of jasmolone and pyrethrolone (Supplemental Figure S3). Second, 184

we derivatized the hydrolyzed extract with MTBSTFA (N-tert-Butyldimethylsilyl-N-185

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methyltrifluoroacetamide) to protect all possible hydroxyl groups from dehydration. By checking 186

the specific ion fragment m/z=194.0, only silylated jasmolone and pyrethrolone were detected. In 187

contrast, no peaks representing single or double silylation products of possible hydroxyl 188

intermediates were detected (Supplemental Figure S4). 189

190

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We then transiently coexpressed the three genes TcJMH, DN144246, and TcGLIP in N. 191

benthamiana leaves fed with both jasmone and chrysanthemic acid. GC-MS analysis of extract 192

of these leaves identified pyrethrin I in addition to jasmolin I (Figure 3, Supplemental Figure S2). 193

Based on these combined results, we designated gene DN144246 as encoding pyrethrolone 194

synthase (TcPYS). TcPYS belongs to the CYP82 family, and was given the official catalog 195

designation CYP82Q3 by Dr. David Nelson (Cytochrome P450 Homepage: 196

https://drnelson.uthsc.edu/CytochromeP450.html) (Nelson et al., 1996). 197

198

To further confirm TcPYS function, we stably coexpressed TcPYS with TcJMH in Arabidopsis, 199

with expression of both genes independently driven by the 35S promoter. Resulting transgenic 200

plants were tested for the production of jasmolone and pyrethrolone (Figure 4). Surprisingly, a 201

small amount of jasmolone, but no pyrethrolone, was detected in extracts from leaves of control 202

plants (i.e., Col-0 non-transgenic plants) fed with 50 µM jasmone (Figure 4). However, plants 203

expressing TcJMH alone contained 7.5-fold greater levels of jasmolone than control plants (and 204

no pyrethrolone), whereas plants expressing both TcJMH and TcPYS contained pyrethrolone in 205

addition to jasmolone (Figure 4). These results are consistent with TcPYS having a desaturase 206

activity on jasmolone. 207

208

Biochemical Characterization of TcPYS 209

210

Microsomes of N. benthamiana leaves expressing TcPYS were prepared and tested for activity 211

with purified jasmolone prepared from hydrolyzed jasmolin I as previously described (Li et al., 212

2018). After incubation overnight, the reaction solutions were extracted with MTBE and the 213

extract analyzed by GC-MS. This analysis detected pyrethrolone as the sole product (Figure 5). 214

By incubating microsomes with different concentrations of jasmolone for 2 h, the Km value for 215

TcPYS with jasmolone was calculated to be 34.3 ± 2.6 µM (means ± SD, n = 3) (Supplemental 216

Figure S5). To test the substrate specificity of TcPYS, we selected several available chemicals 217

with similar structures, including jasmone, MeJA, 4-hydroxy-4-methyl-7-cis-decenoic acid γ-218

lactone, cis-3-Hexenyl 3-methylbutanoate, cis-3-hexenyl benzoate, and dihydrojasmone. TcPYS 219

microsomal preparations did not catalyze the oxidation of any of these compounds, even after 220

overnight incubations (Figure 5). 221

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222

Subcellular Localization of TcPYS 223

224

Since TcJMH was previously localized to the ER, we checked the subcellular localization of 225

TcPYS by fusing the open reading frame of TcPYS with GFP and transiently expressing this 226

construct in Arabidopsis protoplasts under the control of the 35S promoter. Images obtained via 227

confocal microscopy showed the green signal to be associated with the endoplasmic reticulum, 228

as indicated by the overlap with the coexpressed ER marker AtWAK2-mCherry (Zhou et al., 229

2017) (Figure 6). 230

231

Tissue-specific Expression of TcPYS 232

233

We previously showed that TcJMH converts jasmone to jasmolone in the trichomes of the 234

ovaries (Li et al., 2018). To determine the tissue-specific expression pattern of TcPYS, reverse 235

transcription quantitative PCR (RT-qPCR) experiments were performed with transcripts obtained 236

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from five floral developmental stages (Xu et al., 2018) as well from leaf, stem, and root tissues. 237

Disk and ray florets were separated in stage 4 and 5 florets. The transcript analysis showed that 238

TcPYS had a similar expression pattern to that of TcCDS in flowers, with the transcription levels 239

peaking at stages 2 and 3, and transcript levels being barely detectable in leaf, stem, or roots 240

(Figure 7A). 241

242

To further identify in which parts of the flower TcPYS is expressed, we extracted RNA from 243

stage 3 flower trichomes, ovaries, and corollas from disc florets and the entire ray florets for RT-244

qPCR analysis. RT-qPCR experiments indicated that TcPYS transcripts are found almost 245

exclusively in the trichomes (Figure 7B). 246

247

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As our previous work showed that MeJA treatment of leaves induces the transcription of genes 248

involved in pyrethrin biosynthesis (Li et al., 2018), we tested TcPYS for its inducibility by MeJA. 249

RT-qPCR experiments on RNA collected from pyrethrum leaves treated with MeJA indeed 250

showed a characteristic induction of TcPYS by MeJA, with transcript increases peaking at 6–12 h 251

after MeJA application and decreases observed at 24 h (Figure 7C). 252

253

Phylogenetic Tree of Cytochrome P450 CYP82 Family 254

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255

Phylogenetic analysis indicated that TcPYS belongs to the CYP82 clade (Figure 8), and thus it 256

was named accordingly as CYP82Q3. TcPYS is the first protein in the CYP82Q subfamily that 257

has been biochemically characterized. TcPYS shares 61.7% sequence identity with its closest 258

relative, CYP82Q1 from Stevia rebaudiana, a protein that has not yet been functionally 259

characterized. More distantly related, but functionally characterized, proteins in the CYP82 clade 260

act on flavonoids, terpenes, alkaloids, and coumarins (Siminszky et al., 2005; Kruse et al., 2008; 261

Lee et al., 2010; Beaudoin and Facchini, 2013; Winzer et al., 2015; Hori et al., 2018; Tian et al., 262

2018) (Figure 8). 263

264

265

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Discussion 266

267

Coexpression Analysis, Heterologous Expression, and In vitro Biochemical Assays Identify 268

Pyrethrum CYP82Q3 as Pyrethrolone Synthase 269

270

Our previous work suggested that jasmolone is produced in a one-step enzymatic reaction from 271

jasmone, and that pyrethrolone is probably produced from jasmone in a multistep pathway, likely 272

including jasmolone as an intermediate (Li et al., 2018). Here, we employed the N. benthamiana 273

heterologous expression system to coexpress TcJMH, the CYP protein that converts jasmone to 274

jasmolone, together with candidate CYP genes identified by coexpression analysis. This led to 275

identification of TcPYS, which encodes an enzyme that converts jasmolone to pyrethrolone. N. 276

benthamiana leaves coexpressing TcJMH and TcPYS were shown to produce pyrethrolone upon 277

feeding with jasmone. TcPYS is CYP82Q3, a member of the plant-specific CYP82 clade, which 278

includes proteins known to catalyze the modification of other terpenes as well as flavonoids, 279

coumarins, and alkaloids – all plant-specific specialized compounds. Additional evidence for the 280

enzymatic activity of TcPYS in converting jasmolone to pyrethrolone was obtained by 281

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coexpression of TcJMH and TcPYS in transgenic Arabidopsis as well as by in vitro assays of N. 282

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benthamiana microsomes expressing both genes with jasmolone as a substrate. The in vitro 283

assays of TcPYS with jasmolone and related compounds also indicated that the ER-localized 284

enzyme is highly specific for jasmolone. Finally, N. benthamiana leaves coexpressing TcJMH, 285

TcPYS, and TcGLIP produced pyrethrin I upon being fed both jasmone and chrysanthemic acid. 286

287

TcPYS May Act as a Desaturase 288

289

The oxidative conversion of jasmolone to pyrethrolone could proceed via a hydroxyl 290

intermediate followed by dehydration or directly by the removal of two electrons and two 291

protons (Figure 1). The various analyses of the products of the in vitro reaction (Figures 3, 4 and 292

5, and Supplemental Figures S3 and S4) identified only pyrethrolone. Whereas it is not possible 293

to rule out production of short-lived, hard-to-detect hydroxylated intermediates and an 294

endogenous N. benthamiana enzyme that acts on such an intermediate, these results suggest that 295

TcPYS catalyzes the direct formation of a C-C double bond between the terminal carbons of the 296

jasmolone pentyl side chain. If so, it would be the first characterized member of the CYP82 297

subfamily that acts as a desaturase. Most of these proteins catalyze hydroxylation reactions, with 298

some exceptions: CYP82N2/N4 catalyzes a cyclization (Beaudoin and Facchini, 2013; Fujiwara 299

and Ito, 2017), CYP82Y2 catalyzes a regiospecific isomerization (Winzer et al., 2015), 300

CYP82G1 catalyzes oxidative degradation, and CYP82E4v1 catalyzes demethylation (Siminszky 301

et al., 2005; Lee et al., 2010). 302

303

Direct desaturation catalyzed by CYP P450 enzymes is uncommon. In most reactions catalyzed 304

by these enzymes, the activated enzyme-bound oxygen-iron complex ([FeO]3+) attacks a carbon 305

(or N or S) atom and abstracts a hydrogen from it, forming a paired [FeOH]3+ - carbon radical. 306

This is followed by the newly formed hydroxyl rebounding to the carbon, generating a C-OH 307

group. In some rare cases, the abstraction of a hydrogen might be followed by the abstraction of 308

a second hydrogen from an adjacent carbon atom, followed by the formation of a double bond 309

between two atoms, along with the release of a water molecule (Guengerich, 2001). One such 310

example is desaturation at C22 of sterols in fungi and plants, which is catalyzed by CYP61 and 311

CYP710 enzymes, respectively (Kelly et al., 1997; Morikawa et al., 2006). However, C22 of 312

sterols, while on a side chain, is not a terminal or penultimate carbon. The direct desaturation of 313

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valproic acid catalyzed by rat CYP3A1 is a more similar reaction to the formation of the double 314

bond between the ω1-carbon and the ω2-carbon on the side chain of jasmolone by TcPYS 315

(Fisher et al., 1998). Note that in valproic acid oxidation, both hydroxylation and desaturation 316

happen when the initial attack by the enzyme occurs at ω2-carbon, but when the initial attack 317

occurs on the ω1-carbon, the result is 100% hydroxylation (Fisher et al., 1998). 318

319

Pyrethrin Biosynthesis Is a Multi-organellar and Multi-cellular Process 320

321

Maximal production of pyrethrins occurs in the pyrethrum flower, and more specifically in the 322

developing achenes (McLaughlin, 1973). The ovaries contain trichomes, where chrysanthemic 323

acid, 10-carboxychrysanthemic acid, and at least the alcohol jasmolone are produced (Rivera et 324

al., 2001; Li et al., 2018; Xu et al., 2019). The final methylation reaction in the synthesis of 325

pyrethric acid as well as the ester formation of the pyrethrins occurs mostly in the pericarp of the 326

ovaries, and the assembled pyrethrins accumulate throughout the achenes and the seeds, 327

evidently for protection (Ramirez et al., 2012; Xu et al., 2019). Even in the trichomes, the 328

synthesis of these components is divided into multiple organelles. Chrysanthemol is produced in 329

the plastid, whereas the oxidation steps leading to the monoterpene acids occur in the cytosol 330

(Yang et al., 2014; Xu et al., 2018). Jasmonic acid, the precursor of the rethrolones (i.e., 331

jasmolone, pyrethrolone, and cinerolone), is produced from linolenic acid partly in the plastid, 332

partly in the ER, and partly in the peroxisome, whereas the production of jasmolone from 333

jasmone occurs on the ER (Song et al., 1993; Schaller et al., 2000; Ziegler et al., 2000; Matsuda 334

et al., 2005; Ramirez et al., 2013; Li et al., 2018). Here we show that TcPYS is also 335

predominantly expressed in pyrethrum flowers of developmental stages 2 and 3 as found for 336

other pyrethrin biosynthetic genes such as TcCDS and TcJMH (Figure 7A). Furthermore, TcPYS 337

exhibits a pattern of cellular expression (high levels of transcripts in trichomes) and subcellular 338

protein localization (ER localization) that is very similar to that of the TcJMH gene and its 339

protein which generates jasmolone, the substrate of TcPYS (Figures 6, 7). 340

341

Pyrethrins are also synthesized in pyrethrum leaves, although at a low level compared to that in 342

flowers. However, the biosynthesis of pyrethrins in leaves is induced by wounding as well as by 343

the wounding hormone jasmonate (Ueda and Matsuda, 2011; Kikuta et al., 2012). Here we 344

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showed that TcPYS expression is also jasmonate-inducible (Figure 7C), similar to the expression 345

of other pyrethrin biosynthetic genes (Li et al., 2018). 346

347

Overall, the data presented here suggest that TcPYS may be an unusual cytochrome P450 348

oxidoreductase capable of directly introducing a double bond in the side-chain of jasmolone to 349

produce pyrethrolone. With the recent discovery of TcJMH, the enzyme involved in the 350

synthesis of jasmolone from jasmone, and the set of enzymes involved in the conversion of 351

chrysanthemol to chrysanthemic acid and pyrethric acid, only the synthesis of cinerolone 352

remains unsolved in the biosynthetic pathway of pyrethrins. 353

354

355

Materials and Methods 356

357

Plant Growth Condition 358

Pyrethrum (Tanacetum cineraraiifolium) plants were grown on soil in growth chambers under 359

the following conditions: 25°C, 16 h, 400 µmol m−2 s−1 light and 20°C, 8 h dark. Nicotiana 360

benthamiana and Arabidopsis (Arabidopsis thaliana) plants were grown on soil in growth 361

chambers at 22°C, 16 h, 150 µmol m−2 s−1 light and 20°C, 8 h dark in a growth room. 362

363

Transient Expression in N. benthamiana and Jasmone Feeding 364

Cytochrome P450 oxidoreductase gene candidates were inserted into vector pEAQ-HT and 365

mobilized into Agrobacterium strain GV3101 as described in our previous work (Li et al., 2018). 366

Agrobacterium cells were cultured in LB media overnight until the OD value reached 1.0, then 367

collected by centrifugation and resuspended in equal volume of MMA buffer containing 10 mM 368

MES (pH 6.8), 10 mM MgCl2 and 100 µM acetosyringone. Agrobacterium cells were injected 369

into 4-week-old N. benthamiana leaves via the abaxial surface. When performing coexpression 370

of several genes, equal volumes of suspensions of different Agrobacterium cells were mixed 371

together for injection. After 3 days, the leaves were harvested for feeding experiment or 372

preparation of microsomes. 373

374

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In chemical feeding, two N. benthamiana leaves were immersed in 20 mL 2 mM jasmone, placed 375

under vacuum at 12.5 psi for 10 mins, and incubated for 24 h at room temperature. After 376

incubation, leaves were rinsed twice with water, blotted dry with filter paper, and the leaves put 377

into a 15-mL centrifuge tube. MTBE (0.5 mL) was added to the tube and the leaves 378

homogenized with a micro homogenizer to break the tissues. The homogenate was left at room 379

temperature for 1 h, and the sample was centrifuged at 12,000 g for 5 min and the supernatant 380

moved to a 2-mL bottle with a 0.25-mL glass insert for GC-MS analysis. 381

382

For the hydrolysis and derivatization experiments, 36 leaves were first soaked in an aqueous 383

solution containing 2 mM jasmone for 24 h. Afterwards, the leaves were homogenized and 40 ml 384

extract buffer (acetonitrile/ isopropanol/water: 3/3/2) was added and the solution and incubated 385

overnight at 4°C. The solution was next centrifuged at 20000 g for 10 min, and supernatant was 386

lyophilized and resuspended in 1 ml solution of 0.1 M sodium acetate (pH = 5.0). Four mg 387

(≥8 units, one unit liberates 1 μmol of glucose from salicin per min at pH 5.0 at 37°C) of almond 388

β-glucosidase (Sigma-Aldrich G0395), which is known to efficiently hydrolyze β-D-glycoside 389

from a wide range of substrates, were added to 250 µl extract, and incubated for 4 h at 37°C. The 390

lysate was next extracted with 250 µl MTBE for GC-MS analysis. For derivatization, 50 µl 391

hydrolyzed extract was dried in a speed vacuum device, and 100 µl MTBSTFA was added to the 392

dry material which was then incubated at 80°C for 4 h, and analyzed directly on GC-MS. 393

394

Arabidopsis Stable Expression 395

The sequences of TcJMH and TcPYS were inserted into the vector pSAT4A, which contains a 396

CaMV 35S promoter and a CaMV 35S terminator, by double digest with EcoRI and BamHI and 397

ligated with T4 DNA ligase. Next, the complete constructs (promoter-gene-terminator) were 398

obtained by cutting with I-SceI and integrating the fragment into the binary vector pPZP-RSCII. 399

The binary vectors were moved into Agrobacterium GV3101 for Arabidopsis floral dip 400

transformation. TcJMH and TcPYS were transferred into Arabidopsis independently, and the two 401

overexpressing lines were crossed to obtain the coexpression lines. Arabidopsis gene AtActin8 402

was used as internal reference gene to confirm transcription level of TcJMH and TcPYS in 403

transgenic plants. Primers used are listed in Supplemental Table S1. 404

405

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GC-MS Analysis and Km Value Measurement 406

GC-MS analysis was performed on Shimadzu GCMS-QP5000 with a TR-5MS column. The 407

column temperature was programmed as follows: 50°C hold for 2 mins, 50–330°C at 10°C min-1, 408

then hold 10 min. See our previous work for pyrethrolone, jasmolone, jasmolin I and pyrethrin I 409

purification (Li et al., 2018). 410

411

Purification of microsomes was performed using previously published methods (Schaller, 2017). 412

Kinetic parameter measurements were obtained from 50-µL reactions containing 100 mM Tris 413

(pH 7.5), 300 µM NADPH, 40 µl microsome and varying concentration of jasmolone 414

(0/25/50/100/200 µM) or 100 µM substrate for specificity test. After incubation for 2 h (for Km 415

value determination) or overnight (to determine substrate specificity) at room temperature, 416

products were extracted with 100 µl MTBE for GC-MS analysis and their concentrations plotted 417

to verify enzyme saturation. The Km value was calculated by hyperbolic regression analysis 418

method with software Hyper32. Data are presented as means ± SD (n=3). 419

420

RT-qPCR of Floral Transcripts and MeJA Induction of Transcripts in Leaves 421

RNA was extracted with an Omega Plant RNA kit (catalog no. R6827-02). Reverse transcription 422

reaction was carried out with Thermo Fisher high-capacity cDNA reverse transcription kit 423

(catalog no.4368814). RT-qPCR was performed with Thermo Fisher Power SYBR Green PCR 424

master mix (catalog no. 436759). Different stages of Pyrethrum flower were assessed as 425

previously described (Xu et al., 2018). Isolation of floral trichomes was performed using 426

previously published methods (Ramirez et al., 2012) and MeJA induction was done as we 427

previously reported (Li et al., 2018). Transcriptional level of TcPYS was determined by 428

comparison with internal reference genes TcGAPDH, TcActin7 and TcTubulin3. Primers used are 429

listed in Supplemental Table S1. 430

431

432

Subcellular Localization 433

Full-length TcPYS gene sequence was integrated into the expression vector pEZS-NL using the 434

primers listed in Supplemental Table S1. Arabidopsis protoplasts preparation, transformation and 435

confocal microscopy were performed as previously described (Zhou et al., 2017). 436

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437

Phylogenetic Analysis 438

Amino acid sequences of functionally elucidated CYP82 family proteins were downloaded from 439

the National Center for Biotechnology Information (NCBI). A Maximum Likelihood 440

phylogenetic tree was produced by the software program MEGA7, which is based on the JTT 441

matrix model (Kumar et al., 2016). 442

443

Accession numbers 444

Sequence of TcPYS has been submitted to NCBI under the accession number of MG874675. 445

446

Supplemental Data 447

The following supplemental materials are available. 448

Supplemental Figure S1. Comparisons of mass spectra of pyrethrolone generated from different 449

sources. 450

Supplemental Figure S2. Comparisons of mass spectra of pyrethrin I from different sources. 451

Supplemental Figure S3. Analysis of possible glycosylation of rethrolones in N. benthamiana 452

leaves expressing TcJMH and TcPYS. 453

Supplemental Figure S4. GC-MS analysis of derivatized rethrolones in N. benthamiana leaves 454

expressing TcJMH and TcPYS. 455

Supplemental Figure S5. An in vitro enzyme saturation curve for the conversion of jasmolone 456

to pyrethrolone by purified microsome preparations from N. benthamiana leaves expressing 457

TcPYS. 458

Supplemental Table S1. Primers used in this investigation. 459

460

Acknowledgments 461

We thank Dr. David Nelson for naming TcPYS as CYP82Q3. We also thank Dr. A. Daniel Jones 462

and Dr. Anthony Schilmiller from Michigan State University for derivatization suggestions. 463

464

Figure legends 465

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Figure 1. The biosynthetic pathway of pyrethrins as presently known, and proposed step(s) for 466

the synthesis of pyrethrolone in Tanacetum cinerariifolium. The synthesis of jasmonic acid from 467

linolenic acid is catalyzed by lipoxygenase (TcLOX), allene oxidase synthase (TcAOS), allene 468

oxide cyclase (TcAOC), cis (+)-12-oxo-phytodienenic acid reductase (TcOPR) and 3 rounds of 469

β-oxidation. Jasmonic acid is metabolized to jasmone in a set of as yet unidentified reactions. 470

Jasmone is converted to jasmolone in a reaction catalyzed by jasmolone hydroxylase (TcJMH) 471

(Li et al. 2018). Pyrethrolone differs from jasmolone by the presence of a double bond between 472

ω1-carbon and the penultimate carbon of the side chain in the former. The introduction of this 473

double bond in jasmolone could be accomplished by a single reaction or by first hydroxylation 474

and then dehydration. Chrysanthemic acid is generated from two molecules of DMAPP in 475

sequential reactions catalyzed by chrysanthemyl diphosphate synthase/chrysanthemol synthase 476

(TcCDS), alcohol dehydrogenase (TcADH), and aldehyde dehydrogenase (TcALDH) (Xu et al., 477

2018). Pyrethric acid is generated from two molecules of DMAPP by the action of these three 478

enzymes plus chrysanthemol 10-hydroxylase (TcCHH) and 10-carboxychrysanthemicacid 10-479

methyltransferase (TcCCMT) (Xu et al., 2019). The pyrethrin esters are formed in a reaction 480

catalyzed by the GDSL lipase-like protein (TcGLIP), which links the acid moiety to the alcohol 481

moiety (Kikuta et al., 2012). Pyrethrins with chrysanthemic acid as the acid moiety are called 482

type I pyrethrins; pyrethrins with pyrethric acid as the acid moiety are called type II pyrethrins. 483

484

Figure 2. Assay for pyrethrolone synthase activity of the candidate cytochrome P450 485

oxidoreductases identified in our coexpression analysis. The assays were performed by 486

transiently expressing the candidate genes in N. benthamiana for 3 days via agrobacterium 487

infiltration and immersing their leaves in 2 mM jasmone for 24 hours, after which the leaves 488

were ground and extracted with MTBE. Detection of jasmolone and pyrethrolone in the MTBE 489

extracts were by GC-MS (total ion mode). The jasmolone and pyrethrolone standards were 490

obtained as previously described (Li et al., 2018). Pyrethrolone was detected only in leaves 491

coexpressing candidate gene DN144246 with TcJMH. 492

493

Figure 3. Generation of pyrethrin I in leaves of N. benthamiana. Plants transiently coexpressed 494

TcJMH, TcPYS, and TcGLIP for 3 days via agrobacteria infiltration and were immersed in a 495

solution containing 2 mM jasmone and 200 µM Chrysanthemic acid (ChryAcid), after which the 496

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25

leaves were ground and extracted with MTBE. Detection of jasmolone and pyrethrolone in the 497

MTBE extracts was by GC-MS (total ion mode). The jasmolin I and pyrethrin I standards were 498

obtained as described in (Li et al., 2018). Pyrethrin I was detected only in leaves expressing 499

candidate gene DN144246, along with TcJMH and TcGLIP, and only when fed with both 500

chrysanthemic acid and jasmone. 501

502

Figure 4. Coexpression of TcJMH and TcPYS in Arabidopsis. A, GC-MS detection of jasmolone 503

and pyrethrolone in a line stably expressing TcJMH (JMH), two lines expressing TcPYS (line 504

PYS1 and PYS2), and two lines expressing both genes (JMH × PYS1 and JMH × PYS2). Wild 505

type Col-0 line was used as control. B, Reverse transcription quantitative PCR analysis of 506

relative TcJMH and TcPYS transcript levels in lines analyzed in (A). C, Quantification of 507

jasmolone and pyrethrolone levels in the respective lines (N.D., not detected). Data are presented 508

as means ± SD (n = 3). 509

510

Figure 5. In vitro activity and substrate specificity assays for TcPYS. In vitro conversion of 511

jasmolone to pyrethrolone in microsomes prepared from N. benthamiana leaves transiently 512

expressing TcPYS. Substrate specificity was tested on chemicals with similar structures 513

including jasmone, MeJA, hydroxy-4-methyl-7-cis-decenoic acid γ-lactone, cis-3-Hexenyl 3-514

methylbutanoate, cis-3-hexenyl benzoate and dihydrojasmone. The reaction was carried out 515

overnight, after which the solution was extracted with MTBE and the extract analyzed by GC-516

MS. 517

518

Figure 6. Representative results showing subcellular localization of TcPYS. All images are of 519

the same cell. A, Detection of green fluorescence from TcPYS-GFP fusion protein. B, Detection 520

of red fluorescence from the ER marker AtWAK2-mCherry fusion protein. C, Overlay of GFP 521

and red fluorescence from (A) and (B). D, Detection of autofluorescence from chloroplasts. E, 522

Bright field microscopy of the cell under transmitted white light. Bars = 10 µm. 523

524

Figure 7. TcPYS transcription abundance in different tissues and in leaf under MeJA treatment. 525

A, Reverse transcription quantitative PCR analysis of TcPYS transcripts in different 526

developmental stages of the flower (T and B represent ray florets and disk florets, respectively), 527

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26

leaf, stem and root. B, Reverse transcription quantitative PCR analysis of TcPYS transcripts in 528

different parts of stage 3 flowers. C, Reverse transcription quantitative PCR analysis of 2-week-529

old leaves treated with MeJA. Data are presented as means ± SD (n =3 or 4). 530

531

Figure 8. A Maximum Likelihood phylogenetic tree of TcPYS (CYP82Q3) and the proteins 532

most closely related to it from CYP82 family with functional assignments. The tree also includes 533

TcCHH and TcJMH, which are involved in pyrethrin biosynthesis, CYP94B3 which works on 534

jasmonoyl-L-isoleucine and CYP710A1 as representative P450 enzymes with desaturase 535

activities. Sources and functions are as follow: CYP82D62, flavone-6-hydroxylase [Ocimum 536

basilicum]; CYP82D33, flavone-6-hydroxylase [Ocimum basilicum]; CYP82D2, 4'-537

deoxyflavones hydroxylase [Scutellaria baicalensis]; CYP82D1.1, 4'-deoxyflavones hydroxylase 538

[Scutellaria baicalensis]; CYP82D113, 7-keto-δ-cadinene 18-hydroxylase 539

[Gossypium.raimondii]; CYP82Q3, pyrethrolone synthase [Tanacetum cinerariifolium]; 540

CYP82Q1 [Stevia rebaudiana]; CYP82P3, dihydrosanguinarine 10-hydroxylase [Eschscholzia 541

californica subsp. californica]; CYP82P2, dihydrosanguinarine 10-hydroxylase [Eschscholzia 542

californica subsp. californica]; CYP82C4, fraxetin 5-hydroxylase [Arabidopsis thaliana]; 543

CYP82C2, fraxetin 5-hydroxylase [Arabidopsis thaliana]; CYP82N2, protopine 6-544

monooxygenase [Eschscholzia californica]; CYP82Y2, 1,2-dehydroreticulinium reductase 545

[Papaver somniferum]; CYP82N5 N-methystylopine hydroxylase [Eschscholzia californica 546

subsp. californica]; CYP82N4, methyltetrahydroprotoberberine 14-monooxygenase [Papaver 547

somniferum]; CYP82G1, (3E)-4,8-dimethyl-1,3,7-nonatriene synthase [Arabidopsis thaliana]; 548

CYP82E4v1, nicotine demethylase [Nicotiana tabacum]; CYP71BZ1, chrysanthemol 10-549

hydroxylase [Tanacetum cinerariifolium]; CYP71AT148, jasmone hydroxylase [Tanacetum 550

cinerariifolium]; CYP94B3, jasmonoyl-L-isoleucine hydroxylase [Arabidopsis thaliana]; 551

CYP710A1, C22-sterol desaturase [Arabidopsis thaliana]. The tree is drawn to scale (per the 552

number of substitutions per site). 553

554

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27

555

556

557

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