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SCIENCE ADVANCES | RESEARCH ARTICLE BIOSYNTHESIS Copyright © 2020 The Authors, some rights reserved; The evolutionary origins of the cat attractant exclusive licensee American Association nepetalactone in catnip for the Advancement Benjamin R. Lichman1*, Grant T. Godden2, John P. Hamilton3, Lira Palmer4, of Science. No claim to 4 3† 3 3 original U.S. Government Mohamed O. Kamileen , Dongyan Zhao , Brieanne Vaillancourt , Joshua C. Wood , Works. Distributed 2 2,5 6 4 6,7,8 Miao Sun , Taliesin J. Kinser , Laura K. Henry , Carlos Rodriguez-Lopez , Natalia Dudareva , under a Creative 2,5 2 3,9,10 4 Douglas E. Soltis , Pamela S. Soltis , C. Robin Buell *, Sarah E. O’Connor * Commons Attribution NonCommercial Catnip or catmint (Nepeta spp.) is a flowering plant in the mint family (Lamiaceae) famed for its ability to attract cats. License 4.0 (CC BY-NC). This phenomenon is caused by the compound nepetalactone, a volatile iridoid that also repels insects. Iridoids are present in many Lamiaceae species but were lost in the ancestor of the Nepetoideae, the subfamily containing Nepeta. Using comparative genomics, ancestral sequence reconstructions, and phylogenetic analyses, we probed the re-emergence of iridoid biosynthesis in Nepeta. The results of these investigations revealed mechanisms for the loss and subsequent re-evolution of iridoid biosynthesis in the Nepeta lineage. We present evidence for a chronology of events that led to the formation of nepetalactone biosynthesis and its metabolic gene cluster. Downloaded from This study provides insights into the interplay between enzyme and genome evolution in the origins, loss, and re-emergence of plant chemical diversity. INTRODUCTION that Nepeta ISYs are not responsible for determining nepetalactone Plants from the genus Nepeta L. are commonly known as catmint or stereochemistry (7). Instead, ISYs catalyze the reduction of 8OG but http://advances.sciencemag.org/ catnip due to their ability to modify the behavior of cats. Catnip affects do not control the subsequent cyclization. These enzymes yield a approximately two-thirds of domestic cats and many wild felid species reactive intermediate that, without partner enzymes present, spon- including lions, tigers, and ocelots (1) and induces playful actions such taneously cyclizes to form a mixture of products including cis-trans- as rolling over, cheek rubbing, and pawing (2). The responsible agents nepetalactol (8, 9). are nepetalactones, volatile metabolites thought to mimic cat phero- In Nepeta mussinii, several enzymes act in combination with ISY mones (Fig 1A). Most likely, the adaptive function of nepetalactones in to control the formation of nepetalactone stereoisomers (Fig. 1C) (9). Nepeta is to protect against herbivorous insects (3), not to stimulate cats; Three nepetalactol-related short-chain reductase/dehydrogenases notably, nepetalactones can repel insects with efficiencies comparable to (NEPS1, NEPS2, and NEPS3) were identified. NEPS1 is a dehydro- the synthetic repellent N,N-diethyl-meta- toluamide (DEET) (4). Further- genase, catalyzing the formation of cis-trans-nepetalactone or cis-cis- more, Nepeta species produce different nepetalactone stereoisomers, nepetalactone from the corresponding nepetalactol isomer. In with their ratio affecting the effectiveness of insect repellence (4). contrast, NEPS2 and NEPS3 appear to act as cyclases: NEPS2 was found on May 14, 2020 Nepetalactones are iridoids, a class of atypical monoterpenes that to promote the formation of cis-trans-nepetalactol, while NEPS3 act as defensive compounds in some flowering plants. Iridoid bio- promotes the formation of cis-cis-nepetalactol, an isomer only pres- synthesis begins with hydrolysis of geranyl pyrophosphate, catalyzed by ent in trace quantities in the uncatalyzed cyclization. The enzymes geraniol synthase (GES), followed by oxidation, catalyzed by geraniol responsible for the formation of the trans-cis-nepetalactone isomers 8-hydroxylase (G8H) and 8-hydroxygeraniol oxidoreductase (HGO), have not been identified, yet other NEPS homologs are hypothesized yielding the precursor 8-oxogeranial (8OG) (Fig. 1B) (5). The first to play this role. committed step into the iridoid pathway is the reductive cyclization Iridoids are widespread in the asterid subgroup of flowering plants of 8OG into nepetalactol, catalyzed by iridoid synthase (ISY) (6). including the mint family (Lamiaceae) to which Nepeta belongs We initiated elucidation of the biosynthetic basis of the three major (Fig. 2A) (10). Ancestral state reconstructions indicate that iridoid nepetalactone stereoisomers found in Nepeta species and determined biosynthesis was present in the most recent common ancestor of Lamiaceae (Fig. 2) (11). Most iridoid producers in Lamiaceae produce 1Centre for Novel Agricultural Products, Department of Biology, University of iridoid glycosides that act as a defense against chewing insects [e.g., York, York YO10 5DD, UK. 2Florida Museum of Natural History, University of Florida, harpagide; Fig. 1A] (10, 12). However, the ability to produce iridoids Gainesville, FL 32611, USA. 3Department of Plant Biology, Michigan State University, appears to have been lost in the ancestor of the Nepetoideae lineage 4 612 Wilson Road, East Lansing, MI 48824, USA. Department of Natural Product (a large subclade of mints), and mono- and sesquiterpene volatiles Biosynthesis, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany. 5Department of Biology, University of Florida, Gainesville, FL 32611, USA. 6Depart- usurped them as key defensive compounds (10). However, iridoid ment of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. 7Department biosynthesis re-emerged in one genus of Nepetoideae— Nepeta, of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN primarily in the form of the volatile nepetalactones (Fig. 2) (10). 47907, USA. 8Purdue Center for Plant Biology, Purdue University, West Lafayette, IN 47907, USA. 9Plant Resilience Institute, Michigan State University, 612 Wilson Road, Nepeta therefore has emerged as an important model to investigate East Lansing, MI 48824, USA. 10MSU AgBioResearch, Michigan State University, 446 the loss and subsequent evolutionary re-emergence of a major class West Circle Drive, East Lansing, MI 48824, USA. of defensive compounds. *Corresponding author. Email: [email protected] (B.R.L.); [email protected] To uncover how Nepeta forms nepetalactones and how it re-evolved (C.R.B.); [email protected] (S.E.O.) †Present address: Institute of Biotechnology, Cornell University, 525 Tower Rd. the ability to produce iridoids, we sequenced the genomes of two Ithaca, NY 14853, USA. iridoid-producing species [Nepeta cataria L. and Nepeta mussinii Lichman et al., Sci. Adv. 2020; 6 : eaba0721 13 May 2020 1 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE O OH O A O O O OGlc C H H H H HO H H O O NEPS1 O O O O O 8OG NEPS1 NAD+ NADH NEPS2 H H H NADPH cis-trans cis-trans H H OH ISY nepetalactol nepetalactone HO NADP+ cis-trans cis-cis trans-cis OH O nepetalactone nepetalactone nepetalactone Harpagide OH H H O NEPS1 O Major Nepeta iridoids Typical Lamiaceae iridoid O NEPS3 NAD+ NADH H H cis-cis cis-cis B ? OPP OH OH O OH nepetalactol nepetalactone H GES G8H HGO ISY O OH O Iridoids H H OH O ? O O H Geranyl Geraniol 8-Hydroxy- 8-Oxo Nepetalactol H H pyrophosphate geraniol geranial (8OG) trans-cis trans-cis nepetalactol nepetalactone Downloaded from Fig. 1. Iridoid and nepetalactone biosynthesis. (A) Volatile nepetalactone stereoisomers found in Nepeta. Harpagide is a typical nonvolatile iridoid glucoside found in other iridoid-producing Lamiaceae. (B) Early iridoid biosynthetic pathway in plants: geraniol synthase (GES), geraniol 8-hydroxylase (G8H), 8-hydroxygeraniol oxidoreductase (HGO), and iridoid synthase (ISY). (C) Knowledge of nepetalactone biosynthesis in N. mussinii as reported in Lichman et al. (9). The crossed double bond depiction in the ISY product refers to unknown or undefined stereochemistry. http://advances.sciencemag.org/ βR) cl se (P5 ade A B ucta red β- 5 Prostanthera ne ro Westringia te s e Callicarpa g ro Lamioideae P Lamiaceae Ajugoideae Hyssop Ancestral Scutellarioideae N e iridoid + other subfamilies p biosynthesis e t Ocimeae a Elsholtzieae Nepetoideae on May 14, 2020 Salviinae p Loss of iridoid u o biosynthesis Lycopus r g t Prunella u O Menthinae e d Agastache la c ) Re-emergence Glechoma Y IS of iridoid Hyssopus officinalis ( e s biosynthesis a Nepeta cataria h nt I sy Nepeta mussinii rid d Nepeta oi oi d s Irid ynth ase paralog clade Iridoid-producing Lamiaceae Non–iridoid-producing Lamiaceae Non-Lamiaceae ≥95% support Validated ISYs Fig. 2. Lamiaceae and ISY. (A) Phylogenetic tree of Lamiaceae species as reported in (10); black circles, bootstrap support ≥99%. (B) Cladogram of the PRISE gene family. See fig. S15 for annotated phylogram. Spreng. ex. Henckel (syn. Nepeta racemosa Lam.)] along with the RESULTS closely related, non–iridoid-producing hyssop (Hyssopus officinalis L.). The early iridoid pathway in Nepeta and hyssop This comparative genomic approach, combined with phylogenetic The metabolite profiles of N. cataria, N. mussinii, and H. officinalis analysis and enzymology of reconstructed ancestral biosynthetic were analyzed to determine iridoid content (figs. S1 to S7). The major enzymes, provides experimental evidence for a sequence of molec- volatile components of Nepeta leaves and flowers are nepetalactones, ular and genomic events that led to the re-emergence of iridoids

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