Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis Sungbeom Leea, Somayesadat Badieyanb, David R. Bevanc, Marco Herded, Christiane Gatze, and Dorothea Tholla,1 Departments of aBiological Sciences, bBiological Systems Engineering, and cBiochemistry, Virginia Tech, Blacksburg, VA 24061; dDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824; and eThe Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, D-37073 Göttingen, Germany Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved October 26, 2010 (received for review July 9, 2010) Terpene volatiles play important roles in plant-organism interac- In Arabidopsis, emission of TMTT and other volatiles is in- tions as attractants of pollinators or as defense compounds against duced upon leaf damage by the crucifer-specialists Pieris rapae herbivores. Among the most common plant volatiles are homo- and Plutella xylostella (10, 11). The volatile blend was shown to terpenes, which are often emitted from night-scented flowers and attract the parasitic wasp Cotesia rubecula, which parasitizes from aerial tissues upon herbivore attack. Homoterpene volatiles P. rapae larvae and, therefore, led to increased plant fitness (10, released from herbivore-damaged tissue are thought to contribute 12). Although Arabidopsis leaves release none or negligible to indirect plant defense by attracting natural enemies of pests. amounts of DMNT under natural conditions, olfactometer Moreover, homoterpenes have been demonstrated to induce de- experiments with transgenic Arabidopsis plants, which were con- fensive responses in plant–plant interaction. Although early steps stitutively emitting DMNT and its precursor (E)-nerolidol, in the biosynthesis of homoterpenes have been elucidated, the demonstrated the ability of the two volatile compounds to attract identity of the enzyme responsible for the direct formation of P. persimilis (13). these volatiles has remained unknown. Here, we demonstrate that Homoterpenes may also exert other defensive activities, such as CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the the direct repulsion of aphids (14). Moreover, homoterpene emis- Arabidopsis CYP82 family, is responsible for the breakdown of the sion from Arabidopsis is induced upon infection by Pseudomonas PLANT BIOLOGY E E C20-precursor ( , )-geranyllinalool to the insect-induced C16-homo- syringae DC3000 (15) and after fungal elicitor treatment (11). Fi- E E terpene ( , )-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Re- nally, studies in lima bean revealed the ability of TMTT to induce fi E E combinant CYP82G1 shows narrow substrate speci city for ( , )- the expression of defense genes in plant–plant interactions (5). E geranyllinalool and its C15-analog ( )-nerolidol, which is converted Despite the wide occurrence of homoterpenes in floral odors E to the respective C11-homoterpene ( )-4,8-dimethyl-1,3,7-nona- and volatile blends induced by biotic stress, knowledge of the triene (DMNT). Homology-based modeling and substrate docking biosynthesis of these compounds has been fragmentary. The first support an oxidative bond cleavage of the alcohol substrate via syn committed step in the formation of TMTT is the conversion of the -elimination of the polar head, together with an allylic C-5 hy- central C -diterpene precursor geranylgeranyl diphosphate to the drogen atom. CYP82G1 is constitutively expressed in Arabidopsis 20 fl tertiary alcohol (E,E)-geranyllinalool (Fig. 1). A geranyllinalool stems and in orescences and shows highly coordinated herbivore- synthase (GES) has been identified from Arabidopsis (11) and induced expression with geranyllinalool synthase in leaves depend- terpene synthases catalyzing the analogous conversion of the ing on the F-box protein COI-1. CYP82G1 represents a unique char- C -prenyldiphosphate farnesyl diphosphate to (E)-nerolidol in acterized enzyme in the plant CYP82 family with a function as 15 DMNT formation have been characterized (3, 7). Experiments a DMNT/TMTT homoterpene synthase. with stable-isotope precursors suggested a subsequent oxidative degradation of (E,E)-geranyllinalool and (E)-nerolidol to their floral scent | herbivory | terpene biosynthesis respective homoterpenes (Fig. 1) (16). Analogous biosynthetic pathways involving the oxidative C-C bond cleavage of a tertiary lants interact with the environment by producing a variety of alcohol have been described for the dealkylation of 22-hydroxy- Pchemical compounds. In particular, volatile compounds emit- cholesterol into androstenolone (17) and the formation of the fl ted from owers and vegetative plant tissues serve as attractants for furanocoumarin psoralen from its precursor (+)-marmesin in pollinators or exert defensive activities against herbivores, thereby Apiaceae (Ammi majus) (18). Because these pathways are cata- contributing to plant survival and reproductive success. Among the lyzed by one or two enzymes of the cytochrome P450 mono- most common plant volatiles are the irregular C16-homoterpene oxygenase (P450) family, it was assumed that P450-type enzymes (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and its may catalyze the final degradation steps in homoterpene bio- C11-analog (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), both of synthesis (Fig. 1). fl which are widespread oral odor constituents contributing to the Here, we report that CYP82G1 (At3g25180), encoding a P450 “ fl ” fl white- oral image of night scented owers (1). Moreover, TMTT enzyme of the Arabidopsis thaliana CYP82 family, is responsible for and DMNT are released in response to herbivore attack from the the conversion of (E,E)-geranyllinalool to TMTT. CYP82G1 is foliage of gymnosperms (2) and numerous angiosperms, both expressed constitutively in flowers and coexpressed locally with monocots and dicots (3–7). Several studies have indicated a role of homoterpene volatiles in the attraction of herbivore predators in indirect plant defense. For example, de Boer et al. (8) demon- Author contributions: S.L., S.B., D.R.B., M.H., C.G., and D.T. designed research; S.L., S.B., strated that TMTT influenced the foraging behavior of predatory and M.H. performed research; S.L., S.B., D.R.B., M.H., and D.T. analyzed data; and S.L., S.B., mites when emitted in the presence of other induced volatiles from and D.T. wrote the paper. lima bean leaves infested by spider mites (Tetranychus urticae). In The authors declare no conflict of interest. addition, treatment of lima bean with the terpenoid pathway in- This article is a PNAS Direct Submission. hibitor fosmidomycin, which severely reduced the emission of 1To whom correspondence should be addressed. E-mail: [email protected]. homoterpenes, led to a reduced attraction of the predatory mite This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Phytoseiulus persimilis (9). 1073/pnas.1009975107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009975107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 CYP82G1 Gene Knockout Plants Do Not Produce TMTT and Their OPP OPP Phenotype Is Complemented by the Constitutive Expression of GGPP FPP CYP82G1. To determine the function of CYP82G1 in the forma- GES NES tion of TMTT in vivo, a corresponding gene knockout line, GABI- Kat (GK) 377A01 (21), was analyzed. GK377A01 plants carry a fi OH OH T-DNA insertion in the rst intron of the CYP82G1 gene (Fig. 2A). When leaves of homozygous GK377A01 plants were treated with (E,E)-Geranyllinalool (E)-Nerolidol alamethicin, no CYP82G1 transcript was detected by RT-PCR (Fig. 2B) compared with wild-type plants. In contrast, induction of Oxidave P450 P450 degradaon the GES transcript was found in wild-type and CYP82G1 knockout plants, confirming a successful elicitation by alamethicin (Fig. 2B). In agreement with these results, the CYP82G1 mutant produced geranyllinalool but no TMTT in response to alamethicin treat- TMTT DMNT ment (Fig. 2C and Fig. S1C). Interestingly, emission levels of gera- nyllinalool in alamethicin-treated CYP82G1 knockout plants were Fig. 1. Proposed biosynthetic pathways for the formation of volatile similar to those in wild-type plants and no major accumulation of homoterpenes in plants. GGPP, geranylgeranyl diphosphate; FPP, farnesyl geranyllinalool was observed, suggesting feedback regulatory mech- diphosphate; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; DMNT, 4,8- dimethyl-1,3,7-nonatriene; GES, geranyllinalool synthase; NES, nerolidol anisms in the formation of the TMTT precursor. We also tested synthase; P450, cytochrome P450 monooxygenase. for the formation of TMTT in alamethicin-treated knockout lines of At1g19250 (salk_026163) and two other genes (salk_114795 for At3g55970 and salk_073705 for At5g05600), which showed no ex- GES at the sites of insect feeding damage. We further show that the pression in Pro35S:GES × coi1 plants (Fig. S1A and Table S1). None recombinant CYP82G1 enzyme is able to convert (E)-nerolidol, of the mutants showed disrupted TMTT formation (Fig. S1D). the C15-analog of geranyllinalool, to the respective C11-terpene To complement the GK377A01 phenotype, a 1,548-bp CYP82G1 DMNT and that substrate specificity is dependent on the position cDNA (GenBank NM_113423) was expressed constitutively under of the hydroxyl group and configuration of the prenyl chain. the control of the CaMV 35S promoter in the GK377A01 mutant CYP82G1 is unique as