Pyrethrins are synthesized by the plant pyrethrum (), a chrysanthemum relative. These compounds possess efficient insecticidal properties and are not toxic to humans and most vertebrates. Pyrethrum flowers, and to a smaller extent leaves, synthesize six main types of pyrethrins, which are all esters of a monoterpenoid acid moiety and an alcohol moiety derived from jasmonic acid. Here, we identified and characterized the enzyme responsible for the conversion of jasmone, a derivative of jasmonic acid, to jasmolone. Feeding pyrethrum flowers with jasmone resulted in a 4-fold increase in the concentration of free jasmolone as well as smaller but significant proportional increases in free pyrethrolone and all three type I pyrethrins. We used floral transcriptomic data to identify cytochrome P450 genes whose expression patterns were most highly correlated with that of a key gene in pyrethrin biosynthesis, The candidate genes were screened for jasmone hydroxylase activity through transient expression in leaves fed with jasmone. The expression of only one of these candidate genes produced jasmolone; therefore, this gene was named () and given the CYP designation CYP71AT148. The protein encoded by localized to the endoplasmic reticulum, and microsomal preparations from leaves expressing were capable of catalyzing the hydroxylation of jasmone to jasmolone in vitro, with a value of 53.9 µm was expressed almost exclusively in trichomes of floral ovaries and was induced in leaves by jasmonate.

ORCID IDs: 0000-0001-6777-6565 (M.E.S.); 0000-0001-9917-0656 (G.W.)The glycosylation of nicotinate (NA), a key intermediate of the NAD salvage pathway, occurs widely in land plants. However, the physiological function of NA glycosylation is not well understood in planta, and no gene encoding NA glycosyltransferase has been reported to date. NA glycosylation in Arabidopsis thaliana occurs at either the N-or the O-position of the NA molecule, and O-glucosylation appears to be unique to the Brassicaceae. Using gene-enzyme correlations focused on Family 1 glycosyltransferases (GTs; EC 2.4), we identified and characterized three Arabidopsis GTs, which are likely involved in NA glycosylation. These include one NAOGT (UGT74F2; previously identified as a salicylic acid glycosyltransferases) and two NANGTs (UGT76C4 and UGT76C5). Arabidopsis mutants of UGT74F2 accumulate higher levels of free NA, but not salicylic acid, than that of the wild type, and this inversely correlated with seed germination rates under various abiotic stresses. The germination defect of the ugt74f2-1 mutant could be fully complemented by overexpression of UGT74F2. These observations, together with comprehensive chemical analysis, suggest that NA glycosylation may function to protect plant cells from the toxicity of NA overaccumulation during seed germination. Combined with phylogenetic analysis, our results suggest that NAOGTs arose recently in the Brassicaceae family and may provide a fitness benefit. The multifunctionality of UGT74F2 in Arabidopsis is also investigated and discussed.

Summary In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10‐carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides known as pyrethrins. Pyrethrins are 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 synthesized from jasmone, a degradation product of JA, in a single hydroxylation step catalyzed by jasmone hydroxylase (TcJMH). TcJMH belongs to the CYP71 clade of the cytochrome P450 oxidoreductase family. Here, we used coexpression analysis, heterologous gene 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 to catalyze the direct desaturation of the C1-C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS is highly expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T. cinerariifolium genes involved 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 natural pesticide in a heterologous system.

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