Floral Fragrances. a Day in the Life of a Linalool Molecule
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Plant Species Biology (1999) 14, 95Ð120 NEW PERSPECTIVES IN POLLINATION BIOLOGY: FLORAL FRAGRANCES A day in the life of a linalool molecule: Chemical communication in a plant-pollinator system. Part 1: Linalool biosynthesis in ßowering plants ROBERT A. RAGUSO* and ERAN PICHERSKY *Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA. Department of Biology, University of Michigan, Ann Arbor, MI 48109Ð1048, USA Abstract The monoterpene alcohol, linalool, is present in the ßoral fragrance of diverse plant fami- lies and is attractive to a broad spectrum of pollinators, herbivores and parasitoids. Floral emission of linalool has evolved de novo in the fragrant, moth-pollinated annual Clarkia breweri (Gray) Greene (Onagraceae) through a combination of up-regulation and ectopic expression of its biosynthetic enzyme, linalool synthase (LIS), in conjunction with allo- metric size increases in all ßoral organs. Linalool synthase activity and linalool emissions are 1000-fold lower in a sibling species, C. concinna (Fischer & Meyer) Greene, that is diurnally pollinated. Linalool synthase expression is spatially and temporally regulated during C. breweri ßower development, immediately precedes free linalool emission and is absent from nonßoral tissues. Its activity is highest in the style, but most of the linalool product appears to be converted to the pyranoid and furanoid linalool oxides. The LIS structural gene is a member of the terpene synthase gene family, sharing sequence iden- tity with two discrete classes, represented by limonene synthase (LMS) and copalyl pyrophosphate synthase (CPS). Genetic crosses between C. breweri and C. concinna indi- cate that strong linalool emission segregates as a dominant mendelian trait, whereas the inheritance of linalool oxide formation is more complex, suggesting epistatic biosynthetic pathway interactions. We discuss areas for future research, including comparative studies of linalool biosynthesis in different plant families, entrainment of linalool emission to nocturnal circadian rhythms and the induction of vegetative linalool as an indirect her- bivore defense. Keywords: Clarkia breweri, ßoral scent, hawkmoths, head space, gas chromatographyÐmass spec- trometry, monoterpenoids, Onagraceae, terpene synthase. Received 7 April 1999; accepted 15 April 1999 Introduction plants and their vascular descendants with a pharma- copoeia of UV-screening pigments, growth substances, Flowering plants use diverse, multifunctional biosyn- signal transductants, essential amino acids, membrane thetic pathways to produce a broad spectrum of low lipids, allelopathic agents, fungal elicitors and lignin molecular weight, volatile organic compounds (> 700 (Borg-Karlson et al. 1985; Pellmyr & Thien 1986; Metcalf described structures), which collectively impart char- 1987; Bergstršm 1991; Lichtenthaler et al. 1997). The acteristic fragrances to ßoral and vegetative tissues (see appropriation of volatile secondary metabolites as pol- reviews by Williams 1983; Croteau & Karp 1991; Knudsen linator attractants is thought to represent one of the et al. 1993; Dudareva et al. 1999). Most of these metabolic signal events in the evolutionary proliferation of the pathways are ancient, having provided the earliest land angiosperms (Crepet 1983; Robacker et al. 1988; Pellmyr et al. 1991). Although botanists historically attempted to Correspondence: Robert Raguso classify ßoral odors by their organoleptic qualities, chemi- Email: [email protected] cal afÞnities and pollinator associations (Sprengel 1793; © 1999 Society for the Study of Species Biology 96 R. A. RAGUSO AND E. PICHERSKY Delpino 1874; Kerner 1895; Knuth 1898; Vogel 1954; van What is linalool? der Pijl 1961), progress in understanding ßoral scent as a Organic synthesis of linalool natural phenomenon remained elusive until relatively recently due to a lack of appropriate analytical methods. Linalool (3,7-dimethyl-1,6-octadien-3-ol) is an acyclic The application of gas chromatographyÐmass spectrome- monoterpene alcohol with a sweet, pleasant fragrance try (GC-MS) to the identiÞcation of volatiles trapped from that occurs widely among diverse monocot and dicot the ßoral headspace, combined with advances in volatile families and is one of the most frequently encountered trapping technology, has provided a basis for sens- ßoral scent compounds (Knudsen et al. 1993). Linalool is itive, reproducible analyses of ßoral scent chemistry prized by the ßavor and fragrance industry as a compo- (Bergstršm et al. 1980; Williams 1983; Kaiser 1991; Heath nent of bergamot and lavender essential oils and numer- & Manukian 1994; Agelopoulos & Pickett 1998; Raguso & ous commercial perfumes (Hanneguelle et al. 1992; Ohloff Pellmyr 1998). 1994). Because of the chiral properties of its hydroxylated As a result, there is a growing literature characterizing third carbon, linalool occurs in two enantiomeric forms; the ßoral scent chemistry of many ßowering plant (R )-linalool [> 80% in ho oil (Cinnamomum camphora; Lau- species, exploring spatial and temporal scent variation raceae) and rosewood oil] and (S )-linalool in coriander oil within ßowers (Bergstršm et al. 1995; Schiestl et al. 1997), and many ßoral extracts (Bauer et al. 1990). Traditionally, circadian rhythms in fragrance emission (Altenburger & linalool was obtained from a-or b-pinene (isolated from Matile 1988; Hills 1989; Loughrin et al. 1991), differences turpentine) or other terpenoids via a series of redox trans- in scent chemistry between lineages of related species formations (see Landolt et al. 1994). Most modern syn- (Thien et al. 1975; Whitten & Williams 1992; Dobson et al. theses begin with 2-methyl-2-hepten-6-one and proceed 1997) and the role of fragrance in pollinator attraction via base-catalyzed ethynylation with acetylene to dehy- (Metcalf 1987; Gottsberger & Silberbauer-Gottsberger drolinalool, yielding linalool through hydrogenation of 1991; Dobson 1994; Hossaert-McKey et al. 1994). Never- the triple bond in the presence of a palladiumÐcarbon cat- theless, ßoral scent research remains largely descriptive, alyst (Boelens 1982; Bauer et al. 1990; Fig. 1). Alternative with pivotal unresolved questions ranging from the mo- routes include a Grignard reaction between 2-methyl-2- lecular control of biosynthesis to the selective forces hepten-6-one and vinyl halide (Brud & Danevskii 1971) exerted by discriminating pollinators, widespread and synthesis from prenyl phenyl sulfone through reac- methodological variance between studies, no standardi- tion with isoprene oxide and desulfurization with lithium zed graphical methods for comparing differences in scent in ethylamine (Bauer et al. 1990). chemistry and few attempts to vertically integrate dis- The importance of linalool in perfumery and the indus- coveries across levels of biological organization. trial preparation of vitamins A and E has inspired a large In this review, we explore the current state of ßoral scent body of literature devoted to its organic synthesis and the research in microcosm, by following the fate of a repre- conditions under which it is transformed to other com- sentative scent compound, linalool, from its biosynthesis mercially valuable terpenoid alcohols, acetates and oxides and metabolism within ßoral tissues to its emission from (Godtfredsen et al. 1977; Banthorpe et al. 1978a; Baxter et ßoral organs. Our review is divided into two sections. The al. 1978; Boelens 1982; Cori et al. 1986). Linalool is unsta- Þrst part introduces linalool by presenting a synopsis of its ble under acidic conditions, rearranging to geraniol, nerol organic synthesis and chemical properties and surveying and a-terpineol in various ratios depending upon the the breadth of its occurrence in ßowering plants and other speciÞc enantiomer, pH and reaction temperature used organisms. The second part describes events in the natural (Godtfredsen et al. 1977; Baxter et al. 1978). The conv- biosynthesis of linalool, drawing upon biochemical and ersion of linalool, via oxidation with peracetic acid, to molecular studies in a model plant species, Clarkia breweri (Gray) Greene (Onagraceae), whose powerful ßoral scent is rich in linalool and, in conjunction with other ßoral traits, arose as part of an evolutionary transition from a scentless, bee-pollinated ancestry to pollination by noc- turnal moths (MacSwain et al. 1973; Raguso 1995; Raguso & Pichersky 1995). Whenever possible we focus on mech- anism, evaluating the degree to which our Þndings are applicable to other systems and suggesting avenues for future research. The fate of linalool molecules once they are emitted from ßowers will be addressed in a separate Fig. 1 Organic synthesis of linalool from 2-methyl-2-hepten-6- paper, with an emphasis on olfactory detection by nectar- one via base-catalyzed ethynylation in the presence of a palla- foraging insects. dium catalyst. After Boelens 1982. © 1999 Society for the Study of Species Biology, Plant Species Biology, 14, 95Ð120 A DAY IN THE LIFE OF A LINALOOL MOLECULE 97 its pyranoid and furanoid oxides is of particular in- terest, because of their prominence as ßavor components of papaya (Carica papaya, Caricaceae; Schreier & Winterhalter 1986; Flath et al. 1990), grapes (and wines; Vitis vinißora, Vitaceae; Williams et al. 1980; Strauss et al. 1986) and tea leaves, as is reßected in the Ôtea indexÕ, cal- culated as the ratio of linalool and its oxides to the com- bination of linaloic and geraniolic compounds (Guo et al. 1994; Morita et