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Biosynthesis of t-Anethole in Anise ... - Plant Physiology

Biosynthesis of t-Anethole in Anise ... - Plant Physiology

Biosynthesis of t-Anethole in Anise ... - Plant

Biosynthesis of t-Anethole in Anise: Characterization of t-Anol/Isoeugenol Synthase and an O-Methyltransferase Specific for a C7-C8 Propenyl Side Chain 1[W][OA] Takao Koeduka, Thomas J. Baiga, Joseph P. Noel, and Eran Pichersky* Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (T.K., E.P.); and Howard Hughes Medical Institute, Jack H. Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (T.J.B., J.P.N.) The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl- 14 C]adenosyl-Lmethionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent K m values of 145 mM for coumaryl acetate and 230 mM for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with K m values of 19.3 mM for isoeugenol and 54.5 mM for S-[methyl- 14 C]adenosyl-L-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol. The phenylpropenes are a class of volatile compounds found throughout the gymnosperms and angiosperms. When emitted from flowers, they serve as attractants for pollinators, which detect them through their olfactory systems. In addition, at high concentrations their general toxicity to cells renders them useful as defense compounds, and consequently they are found in vegetative tissues of many plant species, although perhaps due to their toxicity they are typically sequestered in specialized structures or quickly emitted from the plant. For example, eugenol and methylchavicol are synthesized and stored in glandular trichomes on the surface of leaves of sweet basil (Ocimum basilicum, Lamiaceae; Gang et al., 2001; Iijima et al., 2004), and eugenol, isoeugenol, and their meth- 1 This work was supported by the National Science Foundation (grant nos. MCB–0718152 [to E.P.] and MCB–0718064 [to J.P.N.]). T.K. was supported by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. * Corresponding author; e-mail lelx@umich.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eran Pichersky (lelx@umich.edu). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128066 ylated derivatives are synthesized and emitted from flowers of Clarkia breweri (Onagraceae; Wang et al., 1997). Because of the antimicrobial and aromatic properties of the phenypropenes, humans have historically used spices and herbs containing them as pesticides and food preservatives. t-Anethole, a main component of the essential oils of anise (Pimpinella anisum, Apiaceae) seeds, star anise (Illicium anisatum), and fennel (Foeniculum vulgare), has been used as a fumigant against mosquitoes (Chang and Ahn, 2002; Gross et al., 2002). Clove (Syzygium aromaticum) buds, which are rich in eugenol, have been used as a source of spice for millennia (Jirovetz et al., 2006). The many compounds of the phenylpropene class differ from one another in two aspects: the moieties attached to the phenyl ring and the position of the double bond in the propenyl side chain (Fig. 1). For example, t-anol and chavicol both have a para-hydroxy functionality on the phenyl ring, but the double bond in the propenyl side chain in t-anol is between C7 and C8, whereas in chavicol it is between C8 and C9. On the other hand, the position of the double bond in the propenyl side chain is the same (C7 and C8) in t-anethole and methylisoeugenol, but the latter has an additional methoxy functionality at the meta-position of the phenyl ring as compared to t-anethole. We have recently isolated two distinct NADPHdependent enzymes, eugenol synthase (EGS) and 384 Plant Physiology, January 2009, Vol. 149, pp. 384–394, www.plantphysiol.org Ó 2008 American Society of Plant Biologists

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