Floral Volatiles: from Biosynthesis to Function

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Plant, Cell and Environment (2014) 37, 1936–1949 doi: 10.1111/pce.12314

Original Article Floral volatiles: from biosynthesis to function

Joëlle K. Muhlemann, Antje Klempien & Natalia Dudareva

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA

ABSTRACT both attraction of pollinators and defence against florivores and pathogens. Based on their biosynthetic origin, floral Floral volatiles have attracted humans’ attention since antiq- VOCs can be divided into three major classes: terpenoids, uity and have since then permeated many aspects of our lives. phenylpropanoids/benzenoids, and fatty acid derivatives Indeed, they are heavily used in perfumes, cosmetics, flavour- (Fig. 1). In addition, sulphur- and nitrogen-containing com- ings and medicinal applications. However, their primary func- pounds contribute to the attraction of pollinators to flowers tion is to mediate ecological interactions between flowers and by mimicking food or brood sources such as carrion or dung a diverse array of visitors, including pollinators, florivores (Wiens 1978; Faegri & van der Pijl 1979; Jürgens et al. 2006). and pathogens. As such, they ultimately ensure the plants’ However, to date, little is known about the biosynthetic path- reproductive and evolutionary success. To date, over 1700 ways leading to the formation of these compounds. floral volatile organic compounds (VOCs) have been identified. Interestingly, they are derived from only a few biochemi- BIOSYNTHETIC PATHWAYS AND GENES cal networks, which include the terpenoid, phenylpropanoid/ INVOLVED IN THE FORMATION OF benzenoid and fatty acid biosynthetic pathways. These FLORAL VOLATILES pathways are intricately regulated by endogenous and external factors to enable spatially and temporally controlled Biosynthesis of terpenoid compounds emission of floral volatiles, thereby fine-tuning the ecological Terpenoids are the largest class of floral volatiles and interactions facilitated by floral volatiles. In this review, we encompass 556 scent compounds, which are derived from will focus on describing the biosynthetic pathways leading to two common interconvertible five-carbon (C5) precursors: floral VOCs, the regulation of floral volatile emission, as well isopentenyl diphosphate (IPP) and its allylic isomer as biological functions of emitted volatiles. dimethylallyl diphosphate (DMAPP) (McGarvey & Croteau

1995). In plants, these C5 precursors are synthesized from two Key-words: benzenoids; floral scent; florivory; phenylpro- independent and compartmentally separated pathways, the panoids; pollination; regulation; terpenoids; volatile organic mevalonic acid (MVA) and the methylerythritol phosphate compounds. (MEP) pathways, which contribute to terpenoid biosynthesis in a species- and/or organ-specific manner (Vranova et al. INTRODUCTION 2013). The MEP pathway operates in plastids (Hsieh et al. Plants are sessile organisms that need to constantly adapt to 2008) and is mainly responsible for the formation of volatile ∼ ∼ changing environments for their survival and reproduction. mono- (C10) and diterpenes (C20)( 53 and 1% of total floral For this environmental adaptation, plants have evolved a terpenoids, respectively) (Knudsen & Gershenzon 2006), wide array of specialized metabolites, also called plant sec- whereas the MVA pathway is distributed among the cytosol, ondary metabolites or plant natural products. To date, over endoplasmic reticulum and peroxisomes (Simkin et al. 2011; 200 000 specialized metabolites have been described (Dixon Pulido et al. 2012), and gives rise to precursors for volatile ∼ & Strack 2003), out of which approximately 1% corresponds sesquiterpenes (C15)(28% of total floral terpenoids). to floral volatile organic compounds (VOCs) identified in 90 While being compartmentally separated, these isoprenoid different angio- and gymnosperm families (Knudsen et al. biosynthetic pathways are connected via a metabolic ‘cross- 2006). VOCs are lipophilic liquids with low molecular weight talk’ mediated by yet unidentified transporter(s) (Bick & and high vapour pressure at ambient temperatures. Physical Lange 2003; Flügge & Gao 2005). Such connectivity of the properties of these compounds allow them to freely cross pathways allows the MEP pathway, often with a higher cellular membranes and be released into the surrounding carbon flux than the MVA route, to support biosynthesis of environment (Pichersky et al. 2006). Biosynthesis of VOCs cytosolically formed terpenoids as was demonstrated in veg- occurs in all plant organs: roots, stems, leaves, fruits, seeds, as etative tissue (Laule et al. 2003; Ward et al. 2011), fruits well as flowers, which were found to release the highest (Gutensohn et al. 2013) and flowers (Laule et al. 2003; amounts and diversity of VOCs. In contrast to VOCs Dudareva et al. 2005; Ward et al. 2011). Indeed, the MEP released from other plant organs, which are exclusively pathway alone supports sesquiterpene biosynthesis in snap- involved in plant defense, floral VOCs assume functions in dragon flowers (Dudareva et al. 2005). Terpenoid research in flowers has predominantly focused Correspondence: N. Dudareva. E-mail: [email protected] on the isolation and characterization of terpene synthase 1936 © 2014 John Wiley & Sons Ltd Floral volatiles 1937

2000; Rohdich et al. 2003; Guirimand et al. 2012) with several excellent reviews devoted to this subject (McGarvey & Croteau 1995; Chappell 2002; Vranova et al. 2013). In brief, the MVA pathway starts from a stepwise condensation of three molecules of acetyl-CoA and consists of six enzymatic reactions while the MEP pathway begins with the condensation of D-glyceraldehyde 3-phosphate and pyruvate and involves seven enzymatic reactions. Volatile terpenoids are synthesized from prenyl diphosphate precursors, which are produced from condensation of IPP and DMAPP by prenyltransferases. Sequential head-to-tail condensation of two IPP and one DMAPP molecules by farnesyl diphosphate (FPP) synthase in the cytosol leads to the formation of FPP,the precursor for sesquiterpenes (Fig. 2). Head-to-tail condensation of one DMAPP with one IPP molecule in plastids results in geranyl pyrophosphate (GPP) formation, the precursor of monoterpenes, and is cata- Figure 1. Major volatile classes emitted by flowers. Based on their biosynthetic origin, volatiles emitted by flowers can be lysed by the GPP synthase (GPPS) (Fig. 2). This enzyme was grouped into one of the three major volatile classes: terpenoids, found to be heterodimeric in Antirrhinum majus (snap- phenylpropanoids/benzenoid, and fatty acid derivatives. Each dragon) and Clarkia breweri, both of which have a floral scent volatile class is represented by a few typical floral scent compounds. bouquet rich in monoterpene compounds (Tholl et al. 2004). Analyses of tissue-specific, developmental and rhythmic (TPS) genes responsible for the final steps in terpenoid expression of the GPPS small subunit showed positive corre- biosynthesis, while genes and cognate enzymes of the MVA lation between expression and monoterpene emission in and MEP pathways were mainly characterized from vegeta- snapdragon flowers (Tholl et al. 2004), whereas no such corre- tive tissues (Cane 1999; Wise & Croteau 1999; Lange et al. lation was found for the large subunit, suggesting that the

Figure 2. Schematic representation of terpenoid VOC biosynthesis. Synthesis of terpenoid VOCs occurs via the cytosolic mevalonic acid (MVA) and the plastidial methylerythritol phosphate (MEP) pathways, the former giving rise to sesquiterpenes and the latter to monoterpenes, diterpenes and volatile carotenoid derivatives. Crosstalk between both pathways is facilitated by the export of IPP from the plastid to the cytosol. Stacked arrows represent multiple biosynthetic steps. Volatile compounds are highlighted with a yellow background. DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; FPPS, FPP synthase; G3P, glyceraldehyde-3-phosphate; GGPP, geranylgeranyl pyrophosphate; GGPPS, GGPP synthase; GPP, geranyl pyrophosphate; GPPS, GPP synthase; IPP, isopentenyl pyrophosphate; TPS, terpene synthase. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 1938 J. K. Muhlemann et al.

Table 1. List of biosynthetic genes involved in ﬁnal steps of ﬂoral volatile formation

Volatile Gene Species Reference

Monoterpenoids 1,8-Cineole CitMTSL1 Citrus unshiu Shimada et al. 2005 NsCIN Nicotiana suaveolens Roeder et al. 2007 Linalool CbLIS Clarkia breweri Dudareva et al. 1996a AmNES/LIS-1 Antirrhinum majus Nagegowda et al. 2008 TPS10 Arabidopsis thaliana Ginglinger et al. 2013 TPS14 A. thaliana Ginglinger et al. 2013 Myrcene Am1e20 A. majus Dudareva et al. 2003 AmOc15 A. majus Dudareva et al. 2003 AlstroTPS Alstroemeria peruviana Aros et al. 2012 E-(β)-Ocimene Am0e23 A. majus Dudareva et al. 2003 CitMTSL4 C. unshiu Shimada et al. 2005 Sesquiterpenoids α-Farnesene AdAFS1 Actinidia deliciosa Nieuwenhuizen et al. 2009 Germacrene D AdGDS1 A. deliciosa Nieuwenhuizen et al. 2009 VvGerD Vitis vinifera Lucker et al. 2004 FC0592 Rosa hybrida Guterman et al. 2002 Nerolidol AmNES/LIS-2 A. majus Nagegowda et al. 2008 AcNES1 Actinidia chinensis Green et al. 2012 Valencene VvVal V. vinifera Lucker et al. 2004 Benzenoids/phenylpropanoids Benzaldehyde AmBALDH A. majus Long et al. 2009 Benzylacetate CbBEAT C. breweri Dudareva et al. 1998 Benzylbenzoate PhBPBT P. hybrida Boatright et al. 2004 Eugenol PhEGS P. hybrida Koeduka et al. 2006 Isoeugenol PhIGS P. hybrida Koeduka et al. 2006 Isomethyleugenol CbIEMT C. breweri Wang et al. 1997 Methylbenzoate AmBAMT A. majus Murﬁtt et al. 2000 PhBSMT1 P. hybrida Negre et al. 2003 PhBSMT2 P. hybrida Negre et al. 2003 Methyleugenol CbIEMT C. breweri Wang et al. 1997 Phenylacetaldehyde PhPAAS P. hybrida Kaminaga et al. 2006 RhPAAS R. hybrida Farhi et al. 2010 2-Phenylethanol RdPAR R. damascena Chen et al. 2011b Phenylethylbenzoate PhBPBT P. hybrida Boatright et al. 2004 Veratrole SlGOMT1 Silene latifolia Gupta et al. 2012

small subunit is responsible for the regulation of GPP deliciosa), where almost all the sesquiterpenes released and subsequently monoterpene formation. Interestingly, a from flowers are the products of either germacrene D homodimeric GPPS with dual prenyltransferase activity synthase1 (AdGDS1) or α-farnesene synthase1 (AdAFS1) (GPPS and FPP synthase activities) was reported in the orchid (Nieuwenhuizen et al. 2009). In addition, some TPSs exhibit Phalaenopsis bellina and demonstrated to be linked to the substrate promiscuity resulting in formation of different prod- emission of linalool and geraniol (Hsiao et al. 2008). ucts. However, in the case of these TPSs their subcellular FPP and GPP serve as substrates for TPSs and cyclases localization and the availability of a particular substrate deter- (Cane 1999; Wise & Croteau 1999), which in plants are mine the type of product formed (Tholl 2006; Nagegowda responsible for the production of a vast variety of volatile et al. 2008). In addition, the diversity of formed volatile terpenoid compounds (Fig. 2). TPSs are highly diversified terpenoids is not only dependent onTPSs,but is also increased throughout the plant kingdom and form a mid-size gene by enzymes modifying TPS products by hydroxylation, dehy- family (Bohlmann et al. 1998; Chen et al. 2011a), which is drogenation and acylation, which enhance their volatility and comprised of more than 100 genes identified in a variety of olfactory properties (Dudareva et al. 2004). plant species, with one-third being isolated from flowers or To date, multiple flower-specific TPSs have been isolated fruits. Almost half of the known TPSs are capable of synthe- and characterized (see Table 1). They were shown to be sizing multiple products from a single prenyl diphosphate responsible for the formation of the monoterpenes linalool precursor (Degenhardt et al. 2009). For instance, the floral (C. breweri, A. majus and Arabidopsis thaliana) (Dudareva volatile blend of Arabidopsis consists of 20 different et al. 1996a; Nagegowda et al. 2008; Ginglinger et al. 2013), sesquiterpenes, almost all of which are synthesized by only E-(β)-ocimene (A. majus and Citrus unshiu) (Dudareva two sesquiterpene synthases, TPS11 and TPS21 (Tholl et al. et al. 2003; Shimada et al. 2005), myrcene (A. majus and 2005). The same is true for the flowers of kiwifruit (Actinidia Alstroemeria peruviana) (Dudareva et al. 2003; Aros et al. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 Floral volatiles 1939

2012) and 1,8-cineole (Nicotiana suaveolens and C. unshiu) formation from CA involves shortening of the propyl side

(Shimada et al. 2005; Roeder et al. 2007), as well as of the chain by a C2 unit and was shown to proceed via a sesquiterpenes nerolidol (A. majus and Actinidia chinensis) β-oxidative, a non-β-oxidative pathway or a combination of (Nagegowda et al. 2008; Green et al. 2012), α-farnesene both (Boatright et al. 2004; Orlova et al. 2006) (Fig. 3). The (A. deliciosa) (Nieuwenhuizen et al. 2009), germacrene D β-oxidative pathway has only recently been fully elucidated (A. deliciosa, Rosa hybrid and Vitis vinifera) (Guterman et al. in petunia flowers and appears to be analogous to fatty 2002; Lucker et al. 2004; Nieuwenhuizen et al. 2009) and acid catabolism and is localized in peroxisomes. The valencene (V. vinifera) (Lucker et al. 2004). pathway begins with an activation of CA to cinnamoyl-CoA, Besides mono- and sesquiterpenes, certain flowers also followed by hydration, oxidation and cleavage of the β-keto emit irregular terpenoids (C8 to C18).They constitute a minor thioester with subsequent formation of benzoyl-CoA (Van class of floral terpenoids (∼7% of all floral terpenoids), which Moerkercke et al. 2009; Klempien et al. 2012; Qualley et al. are formed via a three-step modification including a 2012). Benzaldehyde acts as the key intermediate in the dioxygenase cleavage, enzymatic transformation and acid- alternative non-β-oxidative pathway and is oxidized to catalysed conversion into volatile compounds (Winterhalter benzoic acid by a NAD+-dependent benzaldehyde dehydro- & Rouseff 2001). Interestingly, the dioxygenase cleavage step genase, which has been isolated and characterized from itself can already result in volatile products, such as α- and snapdragon flowers (Long et al. 2009). However, the enzy- β-ionone, geranylacetone, and pseudoionone, as was found to matic reactions leading to benzaldehyde formation remain be the case in petunia flowers (Simkin et al. 2004). unknown.

Formation of ﬂoral phenylpropanoids (C6-C3), includ- Biosynthesis of phenylpropanoid/ ing (iso)eugenol and methyl(iso)eugenol, shares the benzenoid compounds initial biosynthetic steps with the lignin biosynthetic pathway up to the coniferyl alcohol stage. This monolignol Phenylpropanoids and benzenoids represent the second precursor then undergoes two enzymatic reactions that largest class of plant VOCs (Knudsen et al. 2006) and are eliminate the oxygen functionality at the C9 position. exclusively derived from the aromatic amino acid phenylala- The ﬁrst reaction involves acetylation by an acyltrans- nine (Phe) (Fig. 3), which is synthesized via two alternative ferase from the benzylalcohol acetyl-, anthocyanin- pathways (Maeda et al. 2010, 2011; Maeda & Dudareva O-hydroxycinnamoyl-, hydroxycinnamoyl/benzoyl-CoA: 2012; Yoo et al. 2013). Depending on the structure of their anthranilate-N-hydroxycinnamoyl/benzoyl-,and deacetylvin- carbon skeleton, this class is divided into three subclasses: doline acetyltransferases (BAHD) superfamily as was shown phenylpropanoids (with a C6-C3 backbone), benzenoids (C6- for the formation of coniferyl acetate from coniferyl alcohol in

C1) and phenylpropanoid-related compounds (C6-C2). petunia petals (Dexter et al. 2007). Coniferyl acetate is then Phenylpropanoid-related compounds originate directly converted to the phenylpropanoids eugenol and isoeugenol from Phe and constitute approximately 24% of all des- by eugenol and isoeugenol synthases, respectively,which both cribed phenylpropanoid/benzenoid compounds (Knudsen & belong to the pinoresinol-lariciresinol reductase, isoflavone Gershenzon 2006).So far,only genes and enzymes involved in reductase, and phenylcoumaran benzylic ether reductase the biosynthesis of phenylacetaldehyde and 2-phenylethanol (PIP) family of NADPH-dependent reductases (Koeduka have been isolated and characterized (Kaminaga et al. 2006; et al. 2006, 2008) (Fig. 3). Sakai et al. 2007; Farhi et al. 2010; Chen et al. 2011b; In flowers, the diversity of phenylpropanoid/benzenoid Gutensohn et al. 2011; Hirata et al. 2012) (Fig. 3). In petunia compounds is further increased by modifications such as petals, phenylacetaldehyde is produced via an unusual com- methylation, hydroxylation and acetylation of direct scent bined decarboxylation-amine oxidation reaction catalysed by precursors. These modifications enhance the volatility or phenylacetaldehyde synthase (Kaminaga et al. 2006).In roses, olfactory properties of scent compounds. Methylation reac- however, it is formed via two alternative routes: the first tions are catalysed by either O-methyltransferases or carboxyl involves a phenylacetaldehyde synthase similar to the one methyltransferases. O-methyltransferases were shown to be described in petunia, while the second route employs Phe responsible for the synthesis of a diverse array of benzenoids/ deamination by an aromatic amino acid aminotransferase phenylpropanoids, including veratrole in Silene flowers followed by decarboxylation of the formed phenylpyruvate (Gupta et al. 2012; Akhtar & Pichersky 2013), 3,5- intermediate (Sakai et al. 2007; Farhi et al. 2010; Hirata dimethoxytoluene and 1,3,5-trimethoxybenzene in roses et al. 2012). Further conversion of phenylacetaldehyde to (Lavid et al. 2002; Scalliet et al. 2002), and methyleugenol and 2-phenylethanol is catalysed by a phenylacetaldehyde reduc- isomethyleugenol in Clarkia (Wang & Pichersky 1998). tase as was shown in roses (Sakai et al. 2007; Chen et al. Carboxyl methyltransferases, many of which belong to the 2011b). s-adenosyl-L-methionine: salicylic acid carboxyl methyltrans-

The first committed step in benzenoid (C6-C1) and ferase, s-adenosyl-L-methionine: benzoic acid carboxyl phenylpropanoid (C6-C3) biosynthesis is catalysed by a methyltransferase, and theobromine synthase (SABATH) well-characterized and widely distributed enzyme, L- family (D’Auria et al. 2003), are involved in the biosynthesis phenylalanine ammonia-lyase (PAL), which deaminates of volatile esters like methylbenzoate in snapdragon and Phe to trans-cinnamic acid (CA) and competes with phenyl- petunia flowers (Murfitt et al. 2000; Negre et al. 2003) and acetaldehyde synthase for Phe utilization (Fig. 3). Benzenoid methylsalicylate in Clarkia and petunia (Ross et al. 1999; © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 1940 J. K. Muhlemann et al.

Figure 3. Schematic representation of the VOC phenylpropanoid/benzenoid biosynthetic pathway. Phenylpropanoid/benzenoid VOCs are derived from phenylalanine, which itself is synthesized via the shikimate/phenylalanine biosynthetic pathways. Benzoic acid is the central precursor of various benzenoid VOCs and is synthesized via two biosynthetic routes: the β-oxidative pathway (orange background) and non-β-oxidative route. Stacked arrows indicate multiple enzymatic reactions. Volatile compounds are highlighted with a yellow background. AAAT, aromatic amino acid aminotransferase; BA, benzoic acid; BA-CoA, benzoyl-CoA; BAlc, benzylalcohol; BAld, benzaldehyde; BALDH, benzaldehyde dehydrogenase; BB, benzylbenzoate; BEAT, acetyl-CoA:benzylalcohol acetyltransferase; BPBT, benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase; BSMT, benzoic acid/salicylic acid carboxyl methyltransferase; CA, cinnamic acid; CA-CoA, cinnamoyl-CoA; C4H, cinnamate-4-hydroxylase; CNL, cinnamoyl-CoA ligase; Eug, eugenol; IEug, isoeugenol; KAT, 3-ketoacyl-CoA thiolase; MB, methylbenzoate; 3O3PP-CoA, 3-oxo-3-phenylpropionyl-CoA; PAAS, phenylacetaldehyde synthase; PAL, phenylalanine ammonia-lyase; pCA, p-coumaric acid; PEB, phenylethylbenzoate; PhA, phenylacetaldehyde; Phe, L-phenylalanine; PhEth, 2-phenylethanol; PhPyr, phenylpyruvic acid.

Negre et al. 2003). Enzymes from the BAHD superfamily of Biosynthesis of volatile fatty acid derivatives acyltransferases (D’Auria 2006) were shown to be responsible for the biosynthesis of acetylated scent compounds such as Fatty acid derivatives constitute the third class of flower VOCs, benzylacetate in Clarkia (Dudareva et al. 1998), which derive from the unsaturated C18 fatty acids, linolenic and benzoylbenzoate in Clarkia and petunia (D’Auria et al. 2002; linoleic. Biosynthesis of volatile fatty acid derivatives is initi- Boatright et al. 2004; Orlova et al. 2006), and phenylethyl ated by a stereo-specific oxygenation of the octadecanoid benzoate in petunia flowers (Boatright et al. 2004;Orlova et al. precursors, catalysed by a lipoxygenase (LOX) and leads to 2006). formation of 9- and 13-hydroperoxy intermediates (Schaller © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 Floral volatiles 1941

2001; Feussner & Wasternack 2002). These intermediates can Dudareva et al. 2003; Negre et al. 2003; Nagegowda et al. enter two different branches of the LOX pathway, which 2008; Rodriguez-Saona et al. 2011). Within scent-emitting in turn leads to the formation of volatile compounds. Allene tissues, formation of VOCs is often restricted to specific cell oxide synthase (AOS) exclusively utilizes the 13-hydroperoxy types or layers. In snapdragon flowers, for example, intermediate as substrate, converting it to an unstable epoxide, biosynthesis of the major volatile benzenoid compound which is then subjected to a cyclization followed by a reduction methyl benzoate is restricted to the inner epidermal layer of and a series of cyclization reactions to yield jasmonic acid the upper and lower petal lobes (Kolosova et al. 2001b). (JA). In contrast to the AOS branch, hydroperoxide lyase Similar cell-specific expression of scent biosynthetic genes can convert both types of hydroperoxide fatty acid derivatives was also reported for roses and C. breweri (Dudareva et al. into volatile C6 and C9 aldehydes. These saturated or unsatu- 1996b; Bergougnoux et al. 2007). rated C6 and C9 aldehydes are often substrates for alcohol Rhythmicity of floral scent emission has been shown to dehydrogenases giving rise to volatile alcohols, which can be occur in numerous species and often correlates with the further converted to their esters. These C6 and C9 aldehydes activity of the respective pollinators (see e.g. Raguso et al. and alcohols are commonly referred to as green leaf volatiles, 2003; Dötterl et al. 2005; Effmert et al. 2005; Hoballah et al. as they are usually synthesized in vegetative tissues. However, 2005; Rodriguez-Saona et al. 2011). Rhythmic emission they are also important constituents in the floral volatile allows plants to conserve valuable carbon and energy during bouquet of several plant species such as carnation and wild times of the day when their primary pollinators are inactive. snapdragon (Schade et al. 2001; Suchet et al. 2011). Different modes of rhythmic scent release have been The orchids of the genus Ophrys produce an array of fatty described so far. Diurnal rhythmicity in floral VOC emission acid-derived volatiles as well. Within their bouquet, alkenes was observed in plants pollinated during the day whereas are particularly important mediators in the interaction nocturnally emitting plants are visited by pollinators foraging between orchids and their pollinators. Production of alkenes at night (Kolosova et al. 2001a; Waelti et al. 2008). Interest- requires desaturation of fatty acids, a step that is likely medi- ingly, the total amounts of emitted VOCs do not change over ated by acyl-acyl carrier protein (ACP) desaturases. Two the day/night cycle in Dianthus inoxianus; however, the levels isoforms of a stearoyl-acyl carrier protein (ACP) desaturase of compounds contributing to pollinator attraction vary (SAD), namely SAD1 and SAD2, were identified in Ophrys according to visitor activity (Balao et al. 2011). Rhythmicity sphegodes and O. exaltata. However, only expression of of scent emission is often transcriptionally regulated, simi- SAD2 was positively correlated with the formation of larly to its tissue specificity (see e.g. Kolosova et al. 2001a; alkenes in flowers (Schlüter et al. 2011). OsSAD2 is a func- Hendel-Rahmanim et al. 2007; Nagegowda et al. 2008; tional desaturase capable of producing 18:1Δ9(ω-9) and Nieuwenhuizen et al. 2009), although substrate availability 16:1Δ4(ω-12) fatty acid intermediates from which 9-alkenes for scent biosynthetic enzymes was shown to play a regula- and 12-alkenes could be derived. tory role in the emission of some compounds as well (Kolosova et al. 2001a). In addition to being spatially and rhythmically regulated, REGULATION OF FLORAL VOLATILE EMISSION floral scent emission often changes over the lifespan of Spatial, rhythmic and developmental regulation flowers. Usually, emission levels are highest when flowers are of floral scent emission ready for pollination, that is, when anthers are dehisced, and decrease during senescence. Once pollinated, single flowers Flowers have evolved many complex olfactory and visual change or reduce the level of produced volatiles to prevent guides for pollinator attraction. In order to maximize polli- further visits potentially damaging the flower and to redirect nator attraction, floral scent emission is often restricted to visitors to the remaining unpollinated flowers (Schiestl & particular flower tissues and is developmentally and rhyth- Ayasse 2001; Negre et al. 2003; Muhlemann et al. 2006; mically regulated. Rodriguez-Saona et al. 2011). Developmental regulation of Tissue-specific emission of floral VOCs is a characteristic scent emission occurs at several levels, including orchestrated feature of many species. In general, petals are the primary expression of scent biosynthetic genes (Colquhoun et al. source of floral volatiles, although other tissues (stamens, 2010), enzyme activities (see e.g. Pichersky et al. 1995; pistils, sepals and nectaries) also contribute to the floral Dudareva et al. 2000; Shalit et al. 2003; Boatright et al. 2004; bouquet in certain plant species (Dobson et al. 1990, 1996; Nagegowda et al. 2008) and substrate availability (Dudareva Bergström et al. 1995; Flamini et al. 2003; Dötterl & Jürgens et al. 2000). 2005; Farré-Armengol et al. 2013). Emitted from petals, VOCs often enable long-distance attraction of pollinators, Transcriptional network controlling volatile while VOCs produced in nectaries or pollen signal availabil- emission in flowers ity of food sources. Tissue specificity of scent emission is regulated at the level of scent biosynthetic gene expression Orchestrated formation of volatiles from several independ- and enzyme activity. Indeed, many of the scent biosynthetic ent pathways is not only a function of biochemical properties genes isolated so far show a very specific expression profile, of biosynthetic enzymes, but also requires the involvement of with the highest level found in the scent producing parts of transcription factors (TFs). Indeed, coordinated transcrip- the flower (see e.g. Dudareva et al. 1996a; Murfitt et al. 2000; tional regulation of entire scent biosynthetic networks has © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 1942 J. K. Muhlemann et al. been recently shown (Colquhoun & Clark 2011; Muhlemann timing of volatile emission. These factors include pollination et al. 2012), implying that TFs control scent emission. and interactions with floral antagonists. Successful pollina- Despite their importance, only a few TFs regulating tion leads to a decrease or alterations in floral VOC emission the expression of scent biosynthetic genes have been in a variety of plant species (Tollsten & Bergström 1989; identified to date. TFs controlling the flux through the Tollsten 1993; Schiestl & Ayasse 2001; Negre et al. 2003; Theis phenylpropanoid/benzenoid network have recently been iso- & Raguso 2005; Muhlemann et al. 2006; Rodriguez-Saona lated from petunia flowers. ODORANT1 (ODO1), a R2R3- et al. 2011). Besides reducing carbon loss caused by emission, type MYB TF, is exclusively expressed in petunia petal tissue post-pollination changes in VOCs redirect pollinators to yet and regulates the transcription of a major portion of the unpollinated flowers (Schiestl & Ayasse 2001) or prevent shikimate pathway as well as entry points into both the Phe additional visitation of pollinated flowers by flower antago- (i.e. chorismate mutase) and phenylpropanoid (i.e. PAL) nists (Muhlemann et al. 2006). Post-pollination decrease in branchways (Verdonk et al. 2005). ODO1 was also found to emission was shown to occur within 24–96 h of pollinator activate the promoter of an ABC transporter of unknown visitation (depending on the plant species) and appears to be function localized at the plasma membrane (Van Moerkercke triggered once the pollen tubes reach the ovary (Negre et al. et al. 2012a, 2012b). In petunia flowers, ODO1 is positively 2003). Upon fertilization, different molecular mechanisms regulated by another R2R3-type MYB TF, EMISSION were found to trigger the decrease in scent emission. In OF BENZENOIDS II (EOBII), which also activates the petunia flowers, reduced methylbenzoate emission after pol- promoter of the biosynthetic gene isoeugenol synthase lination is the result of transcriptional down-regulation of the (Spitzer-Rimon et al. 2010; Colquhoun et al. 2011b; Van cognate gene in an ethylene-dependent manner (Negre et al. Moerkercke et al. 2011).The recently identified petunia EOBI 2003). In snapdragon flowers, however, the post-pollination was shown to be a flower-specific R2R3-type TF, which acts decrease in methylbenzoate emission largely depends on downstream of EOBII and upstream of ODO1 (Van reduced S-adenosyl-L-methionine:benzoic acid carboxyl Moerkercke et al. 2011; Spitzer-Rimon et al. 2012). Silencing methyltransferase (BAMT) activity and substrate availability of EOBI expression leads to down-regulation of numerous (Negre et al. 2003). genes in the shikimate pathway (5-enolpyruvylshikimate- Altered volatile emission was also reported in the context 3-phosphate synthase, 3-deoxy-D-arabinoheptulosonate of above- and belowground plant–fungus interactions. Infec- 7-phosphate synthase,chorismate synthase,chorismate mutase, tion of Silene latifolia flowers by the anther smut fungus arogenate dehydratase, and prephenate aminotransferase) Microbotryum violaceum results in decreased total scent as well as downstream scent-related genes (PAL, isoeugenol emission and discrimination against infected flowers by the synthase, and benzoic acid/salicylic acid carboxyl methyl- pollinator (Dötterl et al. 2009). The intensity and chemo- transferase) (Spitzer-Rimon et al. 2012). In contrast to ODO1, diversity of floral scent emission also decrease as a function EOBI and EOBII, the MYB4 TF was found to be a repressor of colonization by arbuscular mycorrhizal fungi in of only a single enzyme in the phenylpropanoid pathway, Polemonium viscosum, suggesting that plant–microbe inter- cinnamate-4-hydroxylase, thus controlling the flux towards actions occurring outside of floral tissues can also modulate phenylpropanoid volatile compounds in petunia flowers flower traits providing an additional layer of external regu- (Colquhoun et al. 2011a). latory mechanism (Becklin et al. 2011). While several TFs regulating the phenylpropanoid/ Herbivore-induced plant volatiles (HIPVs) have been benzenoid network have been isolated and characterized, extensively studied in vegetative tissues. Signal perception transcriptional regulation of the terpenoid pathways remains and transduction mechanisms, as well as kinetics of HIPV elusive. MYC2, a basic helix-loop-helix TF,was recently iden- release, are well characterized in these tissues (Dicke & tified in Arabidopsis inflorescences and shown to activate the Baldwin 2010). However, only few studies have linked expression of two sesquiterpene synthase genes TPS11 and florivory with induction of defence VOCs in flowers. One of TPS21 via the gibberellic and JA signalling pathways (Hong the few known examples of florivore-induced volatile emis- et al. 2012). Despite the identification of several TFs for indi- sion is represented by wild parsnip flowers, which emit higher vidual pathways, master regulators, which orchestrate forma- amounts of octyl esters upon infestation with the parsnip tion of diverse volatile blends and act upstream of multiple webworm (Zangerl & Berenbaum 2009). Likewise, metabolic pathways, are yet to be discovered. Recently, Helicoverpa zea larvae feeding on cotton flower buds induce up-regulation of terpenoid and phenylpropanoid pathways emission of terpenes and fatty acid derivatives (Rose & was achieved by overexpression of the production of antho- Tumlinson 2004). cyanin Pigment1 TF in roses (Zvi et al. 2012). However, it remains unknown whether promoters of genes involved in FLORAL VOLATILES AS MEDIATORS IN terpenoid formation are the natural targets for this TF. BIOTIC INTERACTIONS

Changes in floral volatile emission upon diverse Floral VOCs possess multifaceted functions significantly con- biotic interactions tributing to attraction of pollinators and serving as defence compounds against pathogens and florivores.Although it was In addition to internal regulatory mechanisms, several exter- proposed that floral VOCs first served in protecting repro- nal factors are known to influence composition, quantity and ductive structures against antagonists and only later acquired © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 Floral volatiles 1943 pollinator-attracting capacities (Pellmyr & Thien 1986), their ing plant and animal organic matter, were found to arise latter function has been studied the most. Pollinator attrac- independently via convergent evolution (Jürgens et al. 2013). tion is mostly mediated by benzenoids whereas defence func- These emitted volatiles also represent a characteristic feature tions are predominantly assured by terpenoid and benzenoid of plants pollinated by necrophagous flies and beetles, thus VOCs (Schiestl 2010). showing a clear link between certain pollinator guilds and specific floral scent chemistries. Floral volatiles in pollinator attraction Attempts to correlate floral volatile profiles with pollination syndromes have succeeded for certain plant–pollinator For countless cross-pollinating plant species, mating involves interactions; however, clear-cut predictions of pollinator the movement of pollen from one individual to another. In guilds based on floral scent chemistry still remain unattain- many cases, animals such as insects, birds and mammals sig- able. It was nevertheless established that plant species relying nificantly contribute to pollination by serving as vectors in on moths for pollination predominantly release benzenoid, pollen transfer. Flowers employ a diverse palette of signals to terpenoid and nitrogen-containing compounds (Knudsen & mediate attraction of pollinators to flowers for ensuring suc- Tollsten 1993; Dobson 2006), and that bat-pollinated species cessful reproduction. For pollinators, this multisensory mostly emit sulphur-containing volatiles (von Helversen et al. (visual, olfactory, thermal, electromagnetic) input is essential 2000). While involvement of volatiles in the attraction of to locate food and breeding sites. Over the last decade, hummingbirds remains debated, certain ornithophilous mounting evidence was accumulated for the role of floral plants were found to emit minute amounts of terpenoids and volatiles in plant–pollinator communication. fatty acid derivatives (Knudsen et al. 2004). Furthermore, It has been shown that the information conveyed by floral hummingbirds were shown to display different behaviours volatiles depends on amount, composition and context of (attraction or aversion) depending on the volatiles contained their emission, and elicits distinct behavioural responses in in the nectar of artificial flowers (Kessler & Baldwin 2007; the respective pollinators. Long-distance emission of Kessler et al. 2008). volatiles mainly contributes to guiding pollinators to flowers and is especially important for night-emitting plants where Floral volatiles in flower defence production of volatiles has to be of high intensity to over- come decreased conspicuousness of flowers under low illu- Flowers are generally deficient in physical barriers such as a mination. In fact, the moth-pollinated Petunia axillaris and highly lignified cell wall and/or an impermeable cuticle, S. latifolia emit higher amounts of volatiles than day-emitting making them highly susceptible to pathogens and florivores. bee-pollinated plants within the same genus, like Petunia Moreover, they typically carry higher densities of microor- integrifolia and Silene dioica (Ando et al. 2001; Waelti et al. ganisms than other aerial plant surfaces due to their high 2008). In contrast, volatiles emitted over short distances moisture and nutrient content (Johnson & Stockwell 1998). trigger landing, feeding and reproductive behaviour. Expo- Thus, flowers employ volatiles as an alternative mechanism sure even to a single floral volatile of the host plant S. latifolia that prevents damage to their reproductive structures. elicited landing and feeding behaviour in Hadena bicruris Many VOCs were shown to exhibit antimicrobial and anti- moths (Dötterl et al. 2006) while subjection of nocturnal fungal activities in vitro (Bakkali et al. 2008) or inferred to hawkmoth Manduca sexta to an olfactory stimulus induced have these antimicrobial activities due to tissue-specific proboscis extension (Goyret et al. 2007). Not only volatiles expression patterns (e.g. in nectaries and/or stigmas) of their emitted from flower petals but also pollen odour can contrib- biosynthetic genes (Dudareva et al. 1996a; Chen et al. 2003). ute to pollinator foraging. Indeed, pollen odour artificially However, only few VOCs have been investigated for their added to antherless Rosa rugosa flowers increased frequency role in defence against pathogens. (E)-β-caryophyllene of bumblebees’ pollen-collecting behaviour relative to emitted from stigmas of Arabidopsis flowers was shown to odour- and antherless flowers (Dobson et al. 1999). limit bacterial growth. Indeed, Arabidopsis plants lacking Volatiles emitted from flowers not only advertise food (E)-β-caryophyllene emission displayed denser bacterial availability but also mating and oviposition opportunities. populations on their stigmas and reduced seed weight com- Examples include certain orchids that employ flower scent to pared with wild-type plants, indicating that (E)-β- imitate pheromone blends of female pollinators, thereby trig- caryophyllene acts in the defence against pathogenic bacteria gering copulation attempts of male pollinators with flowers and is important for plant fitness (Huang et al. 2012). VOCs (Schiestl et al. 1999). The dioecious species S. latifolia repre- emitted by Saponaria officinalis petals were shown to inhibit sents another example where floral volatiles provide bacterial growth and suggested to control diversity of bacte- oviposition cues for the females of the nursery moth pollina- rial communities in petals (Junker et al. 2011b). tor H. bicruris, which lay eggs while pollinating the flowers Besides pathogens, florivores also cause substantial (Brantjes 1976; Waelti et al. 2009).To mimic oviposition sites, damage to reproductive tissues. Florivores are detrimental to a number of species within the five plant families (Araceae, plant fitness as they feed on reproductive structures, often Rafflesiaceae, Annonaceae, Apocynaceae and Orchidaceae) displace potential pollinators and alter flower morphology emit sulphur-containing volatile compounds to attract (McCall 2008; Sõber et al. 2010). Similar to green tissue necrophagous, saprophagous and caprophagous insects. volatiles, floral volatiles are capable of deterring insects that Interestingly, these volatiles, typically released by decompos- are detrimental to pollinator visitation and/or feeding on © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 1944 J. K. Muhlemann et al.

flower tissues. Ants are very inefficient pollinators (due to are under directional selection pressure. This diversity of their morphology and mobility), often exhibit aggressive selection mechanisms is predicted to lead to the evolution of behaviour against pollinators and occasionally feed on floral very complex floral volatile profiles. structures. It was shown that many common European ants Nursery pollination systems represent a special case where are deterred by volatiles emitted from temperate flowers insects simultaneously pollinate the flowers and use them as (Willmer et al. 2009) and that inhibition of terpene breeding sites. Flowers in these systems have evolved adap- biosynthesis leads to loss of ant-repellent properties (Junker tive mechanisms to reduce damage caused by developing et al. 2011a).The facultative florivore Metrioptera bicolor dis- larvae that feed on the plant’s reproductive tissues (usually plays a strong aversion to linalool and (E)-β-caryophyllene, seeds) (Dufaÿ & Anstett 2003). In general, damage to seeds both of which are widespread terpenoid constituents of scent is predicted to be proportional to the number of visiting bouquets emitted by flowering species (Junker et al. 2010). pollinators. Therefore, fast cessation of floral advertisement These examples strongly suggest involvement of floral after successful pollination is essential to avoid further volatiles in deterrence against undesirable floral visitors. damage to the plant’s reproductive success. Indeed, a post- pollination decrease specifically in pollinator-attracting Balance between defensive and attractive volatiles was observed in Silene, thereby providing a mecha- functions of floral volatiles nism to prevent further loss of seeds (Muhlemann et al. 2006). Pollinators and florivores navigate the same visual and olfactory landscape to locate host plants. To avoid visitation by EVOLUTION OF FLORAL SCENT florivores while advertising their flowers to pollinators, plants have to balance attracting and deterring functions of floral The evolution of angiosperms has resulted in an immense volatiles. Unbalanced production of volatiles involved in dif- diversity of flower traits such as shape, size, colour and scent. ferent functions may result in negative impacts on plant To date, more than 1700 floral volatile compounds have been fitness, as was shown in the cucurbit Cucurbita pepo where described in over 900 flowering plant species (Knudsen et al. enhancement of floral fragrance led to higher attraction of 2006). Interestingly, the quality and quantity of emitted florivores significantly affecting reproductive success (Theis volatiles are species specific and vary among different popu- & Adler 2012). lations of a given species (Raguso 2008). While much effort Several strategies employed by flowers to optimize these has so far been invested in describing scent composition in functions and therefore maximize reproductive success have various flowering species, the mechanisms driving the evolu- been described so far. In Cirsium arvense, some floral tion and diversification of floral scent remain underexplored. volatiles are responsible for attraction of both pollinators Evolution of floral scent is potentially shaped by two factors and florivores (Theis 2006). In this species, timing of emission that mutually influence each other: (1) genomic changes is fine-tuned to increase likelihood of pollinator rather than allowing catalytic expansion and differential regulation of florivore visitation. Indeed, developmental and diel timing of the enzymatic machinery underlying floral scent formation C. arvense volatile emission was found to be positively cor- and (2) ecological constraints such as pollinator-mediated related with the flowers’ reproductive maturity and peak selection. activity of pollinators, respectively, while negative correlation To date, only a few studies have examined the genetic between diel emission and activity of florivores was observed basis for odour differences between closely related flowering (Theis et al. 2007). Similar to C. arvense, VOCs of petunia species. P. axillaris and P. exserta represent a good example flowers attract both pollinators and florivores. Within the of a closely related species with distinct pollination syn- petunia VOC profile, some compounds specifically control dromes. While P. axillaris flowers are colourless, emit infestation rate by florivores (i.e. isoeugenol and benzenoid compounds and are moth pollinated, P. exserta benzylbenzoate) whereas methylbenzoate, for example, is flowers are red, devoid of scent and attract hummingbirds. involved in pollinator attraction. Indeed, utilization of Analysis of the genetic basis for differences in scent profiles various petunia transgenic lines with down-regulation of dif- between these two species revealed that only two quantita- ferent floral scent biosynthetic genes allowed elucidation of tive trait loci are responsible for the distinct scent pheno- the distinct roles of individual volatile compounds in attrac- types (Klahre et al. 2011). One of these loci maps to the tion of mutualists and deterrence of antagonists (Kessler MYB TF ODO1, which controls flux through the shikimate et al. 2013). A similarly complex picture was found in the pathway and hence the amount of precursors available for cucurbit Cucurbita moschata, where some compounds are benzenoid biosynthesis (Verdonk et al. 2005), while the attracting both pollinators and florivores, while other com- genetic identity of the second locus is presently unknown. pounds are only mediating one type of interaction (Andrews C. breweri and C. concinna provide another example of two et al. 2007). As a consequence of interactions with both closely related scented and non-scented species, which rely on mutualists and antagonists, different types of selection may different pollinators. Although non-scented C. concinna act on the different volatile compounds. Indeed, compounds contains genes responsible for formation of VOCs (i.e. lin- involved in attraction of both mutualists and antagonists will alool, (iso)methyleugenol and benzylacetate), transcriptional more likely be under balancing selection, while compounds and/or post-transcriptional regulatory mechanisms lead to involved in interactions solely with one type of flower visitor lack of their expression in flowers and elimination of floral © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 Floral volatiles 1945 scent (Raguso & Pichersky 1995;Dudareva et al. 1996a;Cseke strate that loss or generation of reproductive isolation can et al. 1998; Nam et al. 1999). Differences in transcriptional occur through relatively simple changes in floral scent pro- levels were also found in the flowers of the orchid genus files. Evolution of reproductive isolation through changes in Ophrys, which emit a blend of fatty acid-derived volatiles floral scent chemistry can occur within very short time (Schiestl et al. 1997, 1999; Schiestl & Ayasse 2002).Within this frames, as was illustrated in Ophrys arachnitiformis x blend, alkenes are the key components for the attraction of O. lupercalis hybrids (Vereecken et al. 2010). Indeed, genera- pollinators to flowers and each Ophrys species emits a unique tion of novel floral volatile profiles in these hybrids led to alkene profile, resulting in reproductive isolation between attraction of a pollinator species that does not pollinate any species through attraction of distinct and highly specific pol- of the parent species (Vereecken et al. 2010), thereby leading linators (Schiestl & Ayasse 2002). O. sphegodes mostly emits to rapid floral isolation. 27:1Δ9 and 27:1Δ12 alkenes, while O. exaltata mainly produces a 25:1Δ7 alkene (Schlüter et al. 2011). Formation of double bonds at positions 9 and 12 of these alkenes requires the CONCLUSIONS AND FUTURE PERSPECTIVES action of a SAD. Differences in transcript levels, as well as Over the last two decades, the field of floral volatile research changes in the tertiary structure of SAD2 between has acquired an ever-increasing amount of knowledge on the O. sphegodes and O. exaltata, were proposed as possible functions and biosynthesis of floral scent. Numerous floral mechanisms underlying the distinct alkene profiles and volatile compounds have been identified to date from nearly reproductive isolation (Schlüter et al. 2011). While large 1000 plant species and their importance in mediating ecologi- intra- and interspecific variations in alkene profiles were cal interactions with floral mutualists and antagonists has detected within the Ophrys genus, only limited genetic vari- been highlighted in many plant species. Recent advances in ation among species and populations was observed with the isolation and characterization of genes and enzymes microsatellite markers (Mant et al. 2005). These findings involved in different scent biosynthetic pathways, as well as in suggest that divergent pollinator-mediated selection rather the elucidation of regulatory networks controlling these than genetic drift explains strong differences in volatile pro- pathways, have also enhanced our understanding of how files. Taken together, the above examples demonstrate that floral volatile compounds are synthesized. Despite recent small genetic variations can have large effects on floral scent progress in floral volatile research, many aspects of floral chemistry and interactions with pollinators. volatile function and biosynthesis remain largely unknown. Besides genetic polymorphisms, selection by pollinators is In particular, we still do not know how the majority of floral also capable of driving floral diversification and specializa- VOCs are synthesized, how their orchestrated emission is tion. Pollinator-mediated selection is constrained by the pol- regulated, and the specific roles of floral VOCs in large plant- linator’s pre-existing preferences and sensory abilities, and mutualist/antagonist interaction webs. We therefore antici- can occur only when the traits under selection are heritable pate that future research efforts will focus on providing and exhibit variation (Schiestl 2010; Schiestl & Dötterl 2012). insights into these specific aspects of floral scent biology and Selection on floral scent by pollinators has been described in allow for development of defensive strategies as well as Penstemon digitalis, which displays marked inter-population enhancement of yields in insect-pollinated plants. variation in its emitted floral VOCs. Quantification of phenotypic selection by pollinators revealed that the monoterpene linalool is a direct target of selection within this scent profile ACKNOWLEDGMENTS (Parachnowitsch et al. 2012). Several studies in other plant This work was supported by grants from the National Science species have also uncovered volatile compounds that are Foundation (MCB-0919987) and USDA-NIFA (2011-67013- important for plant–pollinator interaction and thus serve as 30126) to N.D. potential targets for pollinator-mediated selection (see e.g. Dötterl et al. 2006; Shuttleworth & Johnson 2010; Schiestl et al. 2011). REFERENCES Interestingly, changes in floral scent through external Akhtar T.A. & Pichersky E. (2013) Veratrole biosynthesis in white campion. manipulation (genetic or chemical) or intrinsic processes Plant Physiology 162, 52–62. (mutations or hybridization) were shown to drive pollinator Ando T., Nomura M., Tsukahara J., Watanabe H., Kokubun H., Tsukamoto T., niche shifts, which are a proximate cause of floral and repro- . . . Kitching I.J. (2001) Reproductive isolation in a native population of ductive isolation. Indeed, Shuttleworth & Johnson (2010) Petunia sensu Jussieu (Solanaceae). Annals of Botany 88, 403–413. Andrews E.S., Theis N. & Adler L.S. (2007) Pollinator and herbivore attraction have demonstrated that the presence/absence of sulphur to Cucurbita floral volatiles. Journal of Chemical Ecology 33, 1682–1691. compounds within the bouquets of four Eucomis species Aros D., Gonzalez V., Allemann R.K., Muller C.T., Rosati C. & Rogers H.J. determines whether wasps or flies pollinate individual (2012) Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented species. Furthermore, creation of more similar floral volatile Alstroemeria flowers. Journal of Experimental Botany 63, 2739–2752. profiles between S. dioica and S. latifolia by artificial manipu- Bakkali F., Averbeck S., Averbeck D. & Idaomar M. (2008) Biological effects lation of a single compound resulted in higher interspecific of essential oils-a review. Food and Chemical Toxicology 46, 446–475. pollen transfer (Waelti et al. 2008). Thus, the aforementioned Balao F., Herrera J., Talavera S. & Dotterl S. (2011) Spatial and temporal patterns of floral scent emission in Dianthus inoxianus and electro- studies show that floral scent is an important factor in defin- antennographic responses of its hawkmoth pollinator. Phytochemistry 72, ing and maintaining species boundaries. They also demon- 601–609. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949 1946 J. K. Muhlemann et al.

Becklin K.M., Gamez G., Uelk B., Raguso R.A. & Galen C. (2011) Soil fungal Dicke M. & Baldwin I.T. (2010) The evolutionary context for herbivore- effects on floral signals, rewards, and aboveground interactions in an alpine induced plant volatiles: beyond the ‘cry for help’. Trends in Plant Science 15, pollination web. American Journal of Botany 98, 1299–1308. 167–175. Bergougnoux V., Caissard J.C., Jullien F., Magnard J.L., Scalliet G., Cock J.M., Dixon R.A. & Strack D. (2003) Phytochemistry meets genome analysis, and . . . Baudino S. (2007) Both the adaxial and abaxial epidermal layers of the beyond. Phytochemistry 62, 815–816. rose petal emit volatile scent compounds. Planta 226, 853–866. Dobson H.E.M. (2006) Relationship between floral fragrance composition and Bergström G., Dobson H.E.M. & Groth I. (1995) Spatial fragrance patterns type of pollinator. In Biology of Floral Scent (eds N. Dudareva & E. within the flowers of Ranunculus acris (Ranunculaceae). Plant Systematics Pichersky), pp. 147–198. CRC Press, Boca Raton, FL. and Evolution 195, 221–242. Dobson H.E.M., Bergström G. & Groth I. (1990) Differences in fragrance Bick J.A. & Lange B.M. (2003) Metabolic cross talk between cytosolic and chemistry between flower parts of Rosa rugosa Thunb. (Rosaceae). Israel plastidial pathways of isoprenoid biosynthesis: unidirectional transport of Journal of Botany 39, 143–156. intermediates across the chloroplast envelope membrane. Archives of Bio- Dobson H.E.M., Groth I. & Bergström G. (1996) Pollen advertisement: chemi- chemistry and Biophysics 415, 146–154. cal contrasts between whole-flower and pollen odors. American Journal of Boatright J., Negre F., Chen X.L., Kish C.M., Wood B., Peel G., . . . Dudareva N. Botany 83, 877–885. (2004) Understanding in vivo benzenoid metabolism in petunia petal tissue. Dobson H.E.M., Danielson E.M. & Van Wesep I.D. (1999) Pollen odor chemi- Plant Physiology 135, 1993–2011. cals as modulators of bumble bee foraging on Rosa rugosa Thunb. Bohlmann J., Meyer-Gauen G. & Croteau R. (1998) Plant terpenoid synthases: (Rosaceae). Plant Species Biology 14, 153–166. molecular biology and phylogenetic analysis. Proceedings of the National Dötterl S. & Jürgens A. (2005) Spatial fragrance patterns in flowers of Silene Academy of Sciences of the United States of America 95, 4126–4133. latifolia: lilac compounds as olfactory nectar guides? Plant Systematics and Brantjes N.B.M. (1976) Riddles around pollination of Melandrium album Evolution 255, 99–109. (Mill.) Garcke (Caryophyllaceae) during oviposition by Hadena bicruris Dötterl S., Wolfe L.M. & Jürgens A. (2005) Qualitative and quantitative analy- Hufn. (Noctuidae, Lepidoptera). 2. Proceedings of the Koninklijke ses of flower scent in Silene latifolia. Phytochemistry 66, 203–213. Nederlandse Akademie Van Wetenschappen. Series C. Biological and Medical Dötterl S., Jürgens A., Seifert K., Laube T., Weissbecker B. & Schutz S. (2006) Sciences 79, 127–141. Nursery pollination by a moth in Silene latifolia: the role of odours in Cane D.E. (1999) Sesquiterpene biosynthesis: cyclization mechanisms. In eliciting antennal and behavioural responses. New Phytologist 169, 707–718. Comprehensive Natural Products Chemistry: Isoprenoids Including Dötterl S., Jürgens A., Wolfe L. & Biere A. (2009) Disease status and popu- Carotenoids and Steroids (ed. D.D. Cane), pp. 155–200. Elsevier, Amster- lation origin effects on floral scent: potential consequences for oviposition dam, The Netherlands. and fruit predation in a complex interaction between a plant, fungus, and Chappell J. (2002) The genetics and molecular genetics of terpene and sterol noctuid moth. Journal of Chemical Ecology 35, 307–319. origami. Current Opinion in Plant Biology 5, 151–157. Dudareva N., Cseke L., Blanc V.M. & Pichersky E. (1996a) Evolution of floral Chen F., Tholl D., D’Auria J.C., Farooq A., Pichersky E. & Gershenzon J. scent in Clarkia: novel patterns of S-linalool synthase gene expression in the (2003) Biosynthesis and emission of terpenoid volatiles from Arabidopsis C. breweri flower. The Plant Cell 8, 1137–1148. flowers. The Plant Cell 15, 481–494. Dudareva N., Cseke L., Blanc V.M. & Pichersky E. (1996b) Molecular charac- Chen F., Tholl D., Bohlmann J. & Pichersky E. (2011a) The family of terpene terization and cell type-specific expression of linalool synthase gene from synthases in plants: a mid-size family of genes for specialized metabolism Clarkia. Plant Physiology 111, 815–815. that is highly diversified throughout the kingdom. The Plant Journal 66, Dudareva N., D’Auria J.C., Nam K.H., Raguso R.A. & Pichersky E. (1998) 212–229. Acetyl-CoA:benzylalcohol acetyltransferase-an enzyme involved in floral Chen X.M., Kobayashi H., Sakai M., Hirata H., Asai T., Ohnishi T., . . . scent production in Clarkia breweri. The Plant Journal 14, 297–304. Watanabe N. (2011b) Functional characterization of rose phenylacetaldehyde Dudareva N., Murfitt L.M., Mann C.J., Gorenstein N., Kolosova N., Kish C.M., reductase (PAR), an enzyme involved in the biosynthesis of the scent com- . . . Wood K. (2000) Developmental regulation of methyl benzoate pound 2-phenylethanol. Journal of Plant Physiology 168, 88–95. biosynthesis and emission in snapdragon flowers. The Plant Cell 12, 949–961. Colquhoun T.A. & Clark D.G. (2011) Unraveling the regulation of floral Dudareva N., Martin D., Kish C.M., Kolosova N., Gorenstein N., Faldt J., . . . fragrance biosynthesis. Plant Signaling and Behavior 6, 378–381. Bohlmann J. (2003) (E)-beta-ocimene and myrcene synthase genes of floral Colquhoun T.A., Verdonk J.C., Schimmel B.C., Tieman D.M., Underwood B.A. scent biosynthesis in snapdragon: function and expression of three terpene & Clark D.G. (2010) Petunia floral volatile benzenoid/phenylpropanoid synthase genes of a new terpene synthase subfamily. The Plant Cell 15, genes are regulated in a similar manner. Phytochemistry 71, 158–167. 1227–1241. Colquhoun T.A., Kim J.Y., Wedde A.E., Levin L.A., Schmitt K.C., Schuurink Dudareva N., Pichersky E. & Gershenzon J. (2004) Biochemistry of plant R.C. & Clark D.G. (2011a) PhMYB4 fine-tunes the floral volatile signature volatiles. Plant Physiology 135, 1893–1902. of Petunia x hybrida through PhC4H. Journal of Experimental Botany 62, Dudareva N., Andersson S., Orlova I., Gatto N., Reichelt M., Rhodes D., . . . 1133–1143. Gershenzon J. (2005) The nonmevalonate pathway supports both Colquhoun T.A., Schwieterman M.L., Wedde A.E., Schimmel B.C., Marciniak monoterpene and sesquiterpene formation in snapdragon flowers. Proceed- D.M., Verdonk J.C., . . . Clark D.G. (2011b) EOBII controls flower opening ings of the National Academy of Sciences of the United States of America 102, by functioning as a general transcriptomic switch. Plant Physiology 156, 933–938. 974–984. Dufaÿ M. & Anstett M.-C. (2003) Conflicts between plants and pollinators that Cseke L., Dudareva N. & Pichersky E. (1998) Structure and evolution of reproduce within inflorescences: evolutionary variations on a theme. Oikos linalool synthase. Molecular Biology and Evolution 15, 1491–1498. 100, 3–14. D’Auria J.C. (2006) Acyltransferases in plants: a good time to be BAHD. Effmert U., Grosse J., Röse U.S.R., Ehrig F., Kägi R. & Piechulla B. (2005) Current Opinion in Plant Biology 9, 331–340. Volatile composition, emission pattern, and localization of floral scent emis- D’Auria J.C., Chen F. & Pichersky E. (2002) Characterization of an sion in Mirabilis jalapa (Nyctaginaceae). American Journal of Botany 92, acyltransferase capable of synthesizing benzylbenzoate and other volatile 2–12. esters in flowers and damaged leaves of Clarkia breweri. Plant Physiology Faegri K. & van der Pijl L. (1979) The Principles of Pollination Ecology. 130, 466–476. Pergamon Press, Oxford, UK. D’Auria J.C., Chen F., Pichersky E. & John T.R. (2003) The SABATH Farhi M., Lavie O., Masci T., Hendel-Rahmanim K., Weiss D., Abeliovich H. & family of MTS in Arabidopsis thaliana and other plant species. In Vainstein A. (2010) Identification of rose phenylacetaldehyde synthase by Recent Advances in Phytochemistry (ed. J.T. Romeo), pp. 253–283. Elsevier, functional complementation in yeast. Plant Molecular Biology 72, 235–245. Amsterdam. Farré-Armengol G., Filella I., Llusia J. & Peñuelas J. (2013) Floral volatile Degenhardt J., Köllner T.G. & Gershenzon J. (2009) Monoterpene and organic compounds: between attraction and deterrence of visitors under sesquiterpene synthases and the origin of terpene skeletal diversity in plants. global change. Perspectives in Plant Ecology, Evolution and Systematics 15, Phytochemistry 70, 1621–1637. 56–67. Dexter R., Qualley A., Kish C.M., Ma C.J., Koeduka T., Nagegowda D.A., . . . Feussner I. & Wasternack C. (2002) The lipoxygenase pathway. Annual Review Clark D. (2007) Characterization of a petunia acetyltransferase involved in of Plant Biology 53, 275–297. the biosynthesis of the floral volatile isoeugenol. The Plant Journal 49, Flamini G., Cioni P.L. & Morelli I. (2003) Differences in the fragrances of 265–275. pollen, leaves, and floral parts of garland (Chrysanthemum coronarium) and

composition of the essential oils from flower heads and leaves. Journal of Junker R.R., Loewel C., Gross R., Dötterl S., Keller A. & Blüthgen N. (2011b) Agricultural and Food Chemistry 51, 2267–2271. Composition of epiphytic bacterial communities differs on petals and leaves. Flügge U.I. & Gao W. (2005) Transport of isoprenoid intermediates across Plant Biology 13, 918–924. chloroplast envelope membranes. Plant Biology 7, 91–97. Jürgens A., Dötterl S. & Meve U. (2006) The chemical nature of fetid floral Ginglinger J.F., Boachon B., Hofer R. & Werck-Reichhart D. (2013) Gene odours in stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae). New coexpression analysis reveals complex metabolism of the monoterpene Phytologist 172, 452–468. alcohol linalool in Arabidopsis flowers. The Plant Cell. Jürgens A., Wee S.L., Shuttleworth A. & Johnson S.D. (2013) Chemical Goyret J., Markwell P.M. & Raguso R.A. (2007) The effect of decoupling mimicry of insect oviposition sites: a global analysis of convergence in angio- olfactory and visual stimuli on the foraging behavior of Manduca sexta. sperms. Ecology Letters 16, 1157–1167. Journal of Experimental Biology 210, 1398–1405. Kaminaga Y., Schnepp J., Peel G., Kish C.M., Ben-Nissan G., Weiss D., . . . Green S.A., Chen X., Nieuwenhuizen N.J., Matich A.J., Wang M.Y., Bunn B.J., Dudareva N. (2006) Plant phenylacetaldehyde synthase is a bifunctional . . . Atkinson R.G. (2012) Identification, functional characterization, and homotetrameric enzyme that catalyzes phenylalanine decarboxylation and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in oxidation. Journal of Biological Chemistry 281, 23357–23366. kiwifruit (Actinidia chinensis). Journal of Experimental Botany 63, 1951– Kessler D. & Baldwin I.T. (2007) Making sense of nectar scents: the effects of 1967. nectar secondary metabolites on floral visitors of Nicotiana attenuata. The Guirimand G., Guihur A., Phillips M.A., Oudin A., Glevarec G., Melin C., . . . Plant Journal 49, 840–854. Courdavault V. (2012) A single gene encodes isopentenyl diphosphate Kessler D., Gase K. & Baldwin I.T. (2008) Field experiments with transformed isomerase isoforms targeted to plastids, mitochondria and peroxisomes in plants reveal the sense of floral scents. Science 321, 1200–1202. Catharanthus roseus. Plant Molecular Biology 79, 443–459. Kessler D., Diezel C., Clark D.G., Colquhoun T.A. & Baldwin I.T. (2013) Gupta A.K., Akhtar T.A., Widmer A., Pichersky E. & Schiestl F.P. Petunia flowers solve the defence/apparency dilemma of pollinator attrac- (2012) Identification of white campion (Silene latifolia) guaiacol O- tion by deploying complex floral blends. Ecology Letters 16, 299–306. methyltransferase involved in the biosynthesis of veratrole, a key volatile for Klahre U., Gurba A., Hermann K., Saxenhofer M., Bossolini E., Guerin P.M. & pollinator attraction. BMC Plant Biology 12, 158. Kuhlemeier C. (2011) Pollinator choice in petunia depends on two major Gutensohn M., Klempien A., Kaminaga Y., Nagegowda D.A., Negre-Zakharov genetic loci for floral scent production. Current Biology 21, 730–739. F., Huh J.-H., . . . Dudareva N. (2011) Role of aromatic aldehyde synthase in Klempien A., Kaminaga Y., Qualley A., Nagegowda D.A., Widhalm J.R., wounding/herbivory response and flower scent production in different Orlova I., . . . Dudareva N. (2012) Contribution of CoA ligases to benzenoid Arabidopsis ecotypes. The Plant Journal 66, 591–602. biosynthesis in petunia flowers. The Plant Cell 24, 2015–2030. Gutensohn M., Nagegowda D.A. & Dudareva N. (2013) Involvement of Knudsen J.T. & Gershenzon J. (2006) The chemical diversity of floral scent. In compartmentalization in monoterpene and sesquiterpene biosynthesis in Biology of Floral Scent (eds N. Dudareva & E. Pichersky), pp. 27–52. CRC plants. In Isoprenoid Synthesis in Plants and Microorganisms (eds T.J. Bach Press, Boca Raton, FL. & M. Rohmer), pp. 155–169. Springer, New York, NY, USA. Knudsen J.T. & Tollsten L. (1993) Trends in floral scent chemistry in pollina- Guterman I., Shalit M., Menda N., Piestun D., Dafny-Yelin M., Shalev G., . . . tion syndromes: floral scent composition in moth-pollinated taxa. Botanical Weiss D. (2002) Rose scent: genomics approach to discovering novel floral Journal of the Linnean Society 113, 263–284. fragrance-related genes. The Plant Cell 14, 2325–2338. Knudsen J.T., Tollsten L., Groth I., Bergstrom G. & Raguso R.A. (2004) Trends von Helversen O., Winkler L. & Bestmann H.J. (2000) Sulphur-containing in floral scent chemistry in pollination syndromes: floral scent composition in ‘perfumes’ attract flower-visiting bats. Journal of Comparative Physiology.A, hummingbird-pollinated taxa. Botanical Journal of the Linnean Society 146, Sensory, Neural, and Behavioral Physiology 186, 143–153. 191–199. Hendel-Rahmanim K., Masci T., Vainstein A. & Weiss D. (2007) Knudsen J.T., Eriksson R., Gershenzon J. & Stahl B. (2006) Diversity and Diurnal regulation of scent emission in rose flowers. Planta 226, 1491– distribution of floral scent. Botanical Review 72, 1–120. 1499. Koeduka T., Fridman E., Gang D.R., Vassao D.G., Jackson B.L., Kish C.M., . . . Hirata H., Ohnishib T., Ishidac H., Tomidac K., Sakaic M., Harad M. & Pichersky E. (2006) Eugenol and isoeugenol, characteristic aromatic con- Watanabe N. (2012) Functional characterization of aromatic amino acid stituents of spices, are biosynthesized via reduction of a coniferyl alcohol aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose ester. Proceedings of the National Academy of Sciences of the United States of petal protoplasts. Journal of Plant Physiology 169, 444–451. America 103, 10128–10133. Hoballah M.E., Stuurman J., Turlings T.C., Guerin P.M., Connetable S. & Koeduka T., Louie G.V., Orlova I., Kish C.M., Ibdah M., Wilkerson C.G., . . . Kuhlemeier C. (2005) The composition and timing of flower odour emission Pichersky E. (2008) The multiple phenylpropene synthases in both Clarkia by wild Petunia axillaris coincide with the antennal perception and nocturnal breweri and Petunia hybrida represent two distinct protein lineages. The activity of the pollinator Manduca sexta. Planta 222, 141–150. Plant Journal 54, 362–374. Hong G.J., Xue X.Y., Mao Y.B., Wang L.J. & Chen X.Y. (2012) Arabidopsis Kolosova N., Gorenstein N., Kish C.M. & Dudareva N. (2001a) Regulation of MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase circadian methyl benzoate emission in diurnally and nocturnally emitting gene expression. The Plant Cell 24, 2635–2648. plants. The Plant Cell 13, 2333–2347. Hsiao Y.Y., Jeng M.F., Tsai W.C., Chuang Y.C., Li C.Y., Wu T.S., . . . Chen H.H. Kolosova N., Sherman D., Karlson D. & Dudareva N. (2001b) Cellular and (2008) A novel homodimeric geranyl diphosphate synthase from the orchid subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl Phalaenopsis bellina lacking a DD(X)2-4D motif. The Plant Journal 55, methyltransferase, the enzyme responsible for biosynthesis of the volatile 719–733. ester methylbenzoate in snapdragon flowers. Plant Physiology 126, 956– Hsieh M.H., Chang C.Y., Hsu S.J. & Chen J.J. (2008) Chloroplast localization 964. of methylerythritol 4-phosphate pathway enzymes and regulation of mito- Lange B.M., Rujan T., Martin W. & Croteau R. (2000) Isoprenoid biosynthesis: chondrial genes in IspD and IspE albino mutants in Arabidopsis. Plant the evolution of two ancient and distinct pathways across genomes. Proceed- Molecular Biology 66, 663–673. ings of the National Academy of Sciences of the United States of America 97, Huang M., Sanchez-Moreiras A.M., Abel C., Sohrabi R., Lee S., Gershenzon J. 13172–13177. & Tholl D. (2012) The major volatile organic compound emitted from Laule O., Furholz A., Chang H.S., Zhu T., Wang X., Heifetz P.B., . . . Lange M. Arabidopsis thaliana flowers, the sesquiterpene (E)-beta-caryophyllene, (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid is a defense against a bacterial pathogen. New Phytologist 193, 997– biosynthesis in Arabidopsis thaliana. Proceedings of the National Academy 1008. of Sciences of the United States of America 100, 6866–6871. Johnson K.B. & Stockwell V.O. (1998) Management of fire blight: a case Lavid N., Wang J., Shalit M., Guterman I., Bar E., Beuerle T., . . . Lewinsohn E. study in microbial ecology. Annual Review of Phytopathology 36, 227– (2002) O-methyltransferases involved in the biosynthesis of volatile phe- 248. nolic derivatives in rose petals. Plant Physiology 129, 1899–1907. Junker R.R., Heidinger I.M.M. & Blüthgen N. (2010) Floral scent terpenoids Long M.C., Nagegowda D.A., Kaminaga Y., Ho K.K., Kish C.M., Schnepp J., deter the facultative florivore Metrioptera bicolor (Ensifera, Tettigoniidae, . . . Dudareva N. (2009) Involvement of snapdragon benzaldehyde Decticinae). Journal of Orthoptera Research 19, 69–74. dehydrogenase in benzoic acid biosynthesis. The Plant Journal 59, 256– Junker R.R., Gershenzon J. & Unsicker S.B. (2011a) Floral odor bouquet loses 265. its ant repellent properties after inhibition of terpene biosynthesis. Journal Lucker J., Bowen P. & Bohlmann J. (2004) Vitis vinifera terpenoid cyclases: of Chemical Ecology 37, 1323–1331. functional identification of two sesquiterpene synthase cDNAs encoding

(+)-valencene synthase and (−)-germacrene D synthase and expression of Rodriguez-Saona C., Parra L., Quiroz A. & Isaacs R. (2011) Variation in mono- and sesquiterpene synthases in grapevine flowers and berries. highbush blueberry floral volatile profiles as a function of pollination status, Phytochemistry 65, 2649–2659. cultivar, time of day and flower part: implications for flower visitation by McCall A.C. (2008) Florivory affects pollinator visitation and female fitness in bees. Annals of Botany 107, 1377–1390. Nemophila menziesii. Oecologia 155, 729–737. Roeder S., Hartmann A.M., Effmert U. & Piechulla B. (2007) Regulation of McGarvey D.J. & Croteau R. (1995) Terpenoid metabolism. The Plant Cell 7, simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase 1015–1026. of Nicotiana suaveolens. Plant Molecular Biology 65, 107–124. Maeda H. & Dudareva N. (2012) The shikimate pathway and aromatic amino Rohdich F., Zepeck F., Adam P., Hecht S., Kaiser J., Laupitz R., . . . Arigoni D. acid biosynthesis in plants. Annual Review of Plant Biology 63, 73–105. (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Maeda H., Shasany A.K., Schnepp J., Orlova I., Taguchi G., Cooper B.R., . . . studies on the mechanisms of the reactions catalyzed by IspG and IspH Dudareva N. (2010) RNAi suppression of Arogenate Dehydratase1 reveals protein. Proceedings of the National Academy of Sciences of the United States that phenylalanine is synthesized predominantly via the arogenate pathway of America 100, 1586–1591. in petunia petals. The Plant Cell 22, 832–849. Rose U.S. & Tumlinson J.H. (2004) Volatiles released from cotton plants in Maeda H., Yoo H. & Dudareva N. (2011) Prephenate aminotransferase directs response to Helicoverpa zea feeding damage on cotton flower buds. Planta plant phenylalanine biosynthesis via arogenate. Nature Chemical Biology 7, 218, 824–832. 19–21. Ross J.R., Nam K.H., D’Auria J.C. & Pichersky E. (1999) S-Adenosyl-L- Mant J., Peakall R. & Schiestl F.P.(2005) Does selection on floral odor promote methionine:salicylic acid carboxyl methyltransferase, an enzyme involved differentiation among populations and species of the sexually deceptive in floral scent production and plant defense, represents a new class of orchid genus Ophrys? Evolution 59, 1449–1463. plant methyltransferases. Archives of Biochemistry and Biophysics 367, Muhlemann J.K., Waelti M.O., Widmer A. & Schiestl F.P. (2006) Post- 9–16. pollination changes in floral odor in Silene latifolia: adaptive mechanisms for Sakai M., Hirata H., Sayama H., Sekiguchi K., Itano H., Asai T., . . . Watanabe seed-predator avoidance? Journal of Chemical Ecology 32, 1855–1860. N. (2007) Production of 2-phenylethanol in roses as the dominant floral Muhlemann J.K., Maeda H., Chang C.Y., San Miguel P., Baxter I., Cooper B., scent compound from L-phenylalanine by two key enzymes, a PLP- . . . Dudareva N. (2012) Developmental changes in the metabolic network of dependent decarboxylase and a phenylacetaldehyde reductase. Bioscience, snapdragon flowers. PLoS ONE 7, e40381. Biotechnology, and Biochemistry 71, 2408–2419. Murfitt L.M., Kolosova N., Mann C.J. & Dudareva N. (2000) Purification and Scalliet G., Journot N., Jullien F., Baudino S., Magnard J.L., Channeliere S., . . . characterization of S-adenosyl-L-methionine:benzoic acid carboxyl Hugueney P. (2002) Biosynthesis of the major scent components 3,5- methyltransferase, the enzyme responsible for biosynthesis of the volatile dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose ester methyl benzoate in flowers of Antirrhinum majus. Archives of Bio- O-methyltransferases. FEBS Letters 523, 113–118. chemistry and Biophysics 382, 145–151. Schade F., Legge R.L. & Thompson J.E. (2001) Fragrance volatiles of devel- Nagegowda D.A., Gutensohn M., Wilkerson C.G. & Dudareva N. (2008) Two oping and senescing carnation flowers. Phytochemistry 56, 703–710. nearly identical terpene synthases catalyze the formation of nerolidol and Schaller F. (2001) Enzymes of the biosynthesis of octadecanoid-derived signal- linalool in snapdragon flowers. The Plant Journal 55, 224–239. ling molecules. Journal of Experimental Botany 52, 11–23. Nam K.-H., Dudareva N. & Pichersky E. (1999) Characterization of Schiestl F.P. (2010) The evolution of floral scent and insect chemical commu- benzylalcohol acetyltransferases in scented and non-scented Clarkia species. nication. Ecology Letters 13, 643–656. Plant and Cell Physiology 40, 916–923. Schiestl F.P. & Ayasse M. (2001) Post-pollination emission of a repellent com- Negre F., Kish C.M., Boatright J., Underwood B., Shibuya K., Wagner C., pound in a sexually deceptive orchid: a new mechanism for maximising . . . Dudareva N. (2003) Regulation of methylbenzoate emission after reproductive success? Oecologia 126, 531–534. pollination in snapdragon and petunia flowers. The Plant Cell 15, 2992–3006. Schiestl F.P. & Ayasse M. (2002) Do changes in floral odor cause speciation Nieuwenhuizen N.J., Wang M.Y., Matich A.J., Green S.A., Chen X., Yauk Y.K., in sexually deceptive orchids? Plant Systematics and Evolution 234, 111– . . . Atkinson R.G. (2009) Two terpene synthases are responsible for the 119. major sesquiterpenes emitted from the flowers of kiwifruit (Actinidia Schiestl F.P. & Dötterl S. (2012) The evolution of floral scent and olfactory deliciosa). Journal of Experimental Botany 60, 3203–3219. preferences in pollinators: coevolution or pre-existing bias? Evolution 66, Orlova I., Marshall-Colon A., Schnepp J., Wood B., Varbanova M., Fridman E., 2042–2055. . . . Dudareva N. (2006) Reduction of benzenoid synthesis in petunia flowers Schiestl F.P., Ayasse M., Paulus H.F., Erdmann D. & Francke W. (1997) Vari- reveals multiple pathways to benzoic acid and enhancement in auxin trans- ation of floral scent emission and postpollination changes in individual port. The Plant Cell 18, 3458–3475. flowers of Ophrys sphegodes subsp. sphegodes. Journal of Chemical Ecology Parachnowitsch A.L., Raguso R.A. & Kessler A. (2012) Phenotypic selection 23, 2881–2895. to increase floral scent emission, but not flower size or colour in bee- Schiestl F.P., Ayasse M., Paulus H.F., Löfstedt C., Hansson B.S., Ibarra F. & pollinated Penstemon digitalis. New Phytologist 195, 667–675. Francke W. (1999) Orchid pollination by sexual swindle. Nature 399, 421– Pellmyr O. & Thien L.B. (1986) Insect reproduction and floral fragrances – 422. Keys to the evolution of the angiosperms. Taxon 35, 76–85. Schiestl F.P., Huber F.K. & Gomez J.M. (2011) Phenotypic selection on floral Pichersky E., Lewinsohn E. & Croteau R. (1995) Purification and characteri- scent: trade-off between attraction and deterrence? Evolutionary Ecology zation of S-linalool synthase, an enzyme involved in the production of floral 25, 237–248. scent in Clarkia breweri. Archives of Biochemistry and Biophysics 316, 803– Schlüter P.M.,Xu S., Gagliardini V., Whittle E., Shanklin J., Grossniklaus U. & 807. Schiestl F.P. (2011) Stearoyl-acyl carrier protein desaturases are associated Pichersky E., Noel J.P. & Dudareva N. (2006) Biosynthesis of plant volatiles: with floral isolation in sexually deceptive orchids. Proceedings of the nature’s diversity and ingenuity. Science 311, 808–811. National Academy of Sciences of the United States of America 108, 5696– Pulido P., Perello C. & Rodriguez-Concepcion M. (2012) New insights into 56701. plant isoprenoid metabolism. Molecular Plant 5, 964–967. Shalit M., Guterman I., Volpin H., Bar E., Tamari T., Menda N., . . . Lewinsohn Qualley A.V., Widhalm J.R., Adebesin F., Kish C.M. & Dudareva N. (2012) E. (2003) Volatile ester formation in roses. Identification of an acetyl- Completion of the core β-oxidative pathway of benzoic acid biosynthesis in coenzyme A. Geraniol/citronellol acetyltransferase in developing rose plants. Proceedings of the National Academy of Sciences of the United States petals. Plant Physiology 131, 1868–1876. of America 109, 16383–16388. Shimada T., Endo T., Fujii H., Hara M. & Omura M. (2005) Isolation and Raguso R.A. (2008) Wake up and smell the roses: the ecology and evolution of characterization of (E)-beta-ocimene and 1,8 cineole synthases in Citrus floral scent. Annual Review of Ecology, Evolution, and Systematics 39, 549– unshiu Marc. Plant Science 168, 987–995. 569. Shuttleworth A. & Johnson S.D. (2010) The missing stink: sulphur compounds Raguso R.A. & Pichersky E. (1995) Floral volatiles from Clarkia breweri and can mediate a shift between fly and wasp pollination systems. Proceedings of C. concinna (Onagraceae): recent evolution of floral scent and moth polli- the Royal Society B-Biological Sciences 277, 2811–2819. nation. Plant Systematics and Evolution 194, 55–67. Simkin A.J., Underwood B.A., Auldridge M., Loucas H.M., Shibuya K., Raguso R.A., Levin R.A., Foose S.E., Holmberg M.W. & McDade L.A. (2003) Schmelz E., . . . Klee H.J. (2004) Circadian regulation of the PhCCD1 carot- Fragrance chemistry, nocturnal rhythms and pollination ‘syndromes’ in enoid cleavage dioxygenase controls emission of beta-ionone, a fragrance Nicotiana. Phytochemistry 63, 265–284. volatile of petunia flowers. Plant Physiology 136, 3504–3514.

Simkin A.J., Guirimand G., Papon N., Courdavault V., Thabet I., Ginis O., . . . Van Moerkercke A., Haring M.A. & Schuurink R.C. (2012b) A model for Clastre M. (2011) Peroxisomal localisation of the final steps of the mevalonic combinatorial regulation of the petunia R2R3-MYB transcription factor acid pathway in planta. Planta 234, 903–914. ODORANT1. Plant Signaling and Behavior 7, 518–520. Sõber V., Mooraa M. & Tederb T. (2010) Florivores decrease pollinator visita- Verdonk J.C., Haring M.A., van Tunen A.J. & Schuurink R.C. (2005) tion in a self-incompatible plant. Basic and Applied Ecology 11, 669–675. ODORANT1 regulates fragrance biosynthesis in petunia flowers. The Plant Spitzer-Rimon B., Marhevka E., Barkai O., Marton I., Edelbaum O., Masci T., Cell 17, 1612–1624. . . . Vainstein A. (2010) EOBII, a gene encoding a flower-specific regulator of Vereecken N.J., Cozzolino S. & Schiestl F.P. (2010) Hybrid floral scent novelty phenylpropanoid volatiles’ biosynthesis in petunia. The Plant Cell 22, 1961– drives pollinator shift in sexually deceptive orchids. BMC Evolutionary 1976. Biology 10, 103. Spitzer-Rimon B., Farhi M., Albo B., Cna’ani A., Ben Zvi M.M., Masci T., . . . Vranova E., Coman D. & Gruissem W. (2013) Network analysis of the MVA Vainstein A. (2012) The R2R3-MYB-like regulatory factor EOBI, acting and MEP pathways for isoprenoid synthesis. Annual Review of Plant downstream of EOBII, regulates scent production by activating ODO1 and Biology 64, 665–700. structural scent-related genes in petunia. The Plant Cell 24, 5089–5105. Waelti M.O., Muhlemann J.K., Widmer A. & Schiestl F.P. (2008) Floral odour Suchet C., Dormont L., Schatz B., Giurfa M., Simon V., Raynaud C. & Chave and reproductive isolation in two species of Silene. Journal of Evolutionary J. (2011) Floral scent variation in two Antirrhinum majus subspecies influ- Biology 21, 111–121. ences the choice of naïve bumblebees. Behavioural Ecology and Sociobiol- Waelti M.O., Page P.A., Widmer A. & Schiestl F.P. (2009) How to be an ogy 65, 1015–1027. attractive male: floral dimorphism and attractiveness to pollinators in a Theis N. (2006) Fragrance of Canada thistle (Cirsium arvense) attracts both dioecious plant. BMC Evolutionary Biology 9, 190. floral herbivores and pollinators. Journal of Chemical Ecology 32, 917–927. Wang J. & Pichersky E. (1998) Characterization of S-adenosyl-L- Theis N. & Adler L.S. (2012) Advertising to the enemy: enhanced floral fra- methionine:(iso)eugenol O-methyltransferase involved in floral scent pro- grance increases beetle attraction and reduces plant reproduction. Ecology duction in Clarkia breweri. Archives of Biochemistry and Biophysics 349, 93, 430–435. 153–160. Theis N. & Raguso R.A. (2005) The effect of pollination on floral fragrance in Wang J., Dudareva N., Bhakta S., Raguso R.A. & Pichersky E. (1997) thistles. Journal of Chemical Ecology 31, 2581–2600. Floral scent production in Clarkia breweri (Onagraceae). II. Localiza- Theis N., Lerdau M. & Raguso R.A. (2007) The challenge of attracting polli- tion and developmental modulation of the enzyme S-adenosyl-L- nators while evading floral herbivores: patterns of fragrance emission in methionine:(Iso)eugenol O-methyltransferase and phenylpropanoid emis- Cirsium arvense and Cirsium repandum (Asteraceae). International Journal sion. Plant Physiology 114, 213–221. of Plant Sciences 168, 587–601. Ward J.L., Baker J.M., Llewellyn A.M., Hawkins N.D. & Beale M.H. (2011) Tholl D. (2006) Terpene synthases and the regulation, diversity and bio- Metabolomic analysis of Arabidopsis reveals hemiterpenoid glycosides as logical roles of terpene metabolism. Current Opinion in Plant Biology 9, products of a nitrate ion-regulated, carbon flux overflow. Proceedings of the 297–304. National Academy of Sciences of the United States of America 108, 10762– Tholl D., Kish C.M., Orlova I., Sherman D., Gershenzon J., Pichersky E. & 10767. Dudareva N. (2004) Formation of monoterpenes in Antirrhinum majus and Wiens D. (1978) Mimicry in plants. Evolutionary Biology 11, 365–403. Clarkia breweri flowers involves heterodimeric geranyl diphosphate Willmer P.G., Nuttman C.V., Raine N.E., Stone G.N., Pattrick J.G., Henson K., synthases. The Plant Cell 16, 977–992. . . . Knudsen J.T. (2009) Floral volatiles controlling ant behaviour. Functional Tholl D., Chen F., Petri J., Gershenzon J. & Pichersky E. (2005) Two Ecology 23, 888–900. sesquiterpene synthases are responsible for the complex mixture of Winterhalter P. & Rouseff R. (2001) Carotenoid-derived aroma compounds: sesquiterpenes emitted from Arabidopsis flowers. The Plant Journal 42, an introduction. In Carotenoid-Derived Aroma Compounds (eds 757–771. P. Winterhalter & R. Rouseff), pp. 1–17. American Chemical Society, Tollsten L. (1993) A multivariate approach to postpollination changes in the Washington, DC. floral scent of Platanthera bifolia (Orchidaceae). Nordic Journal Of Botany Wise M.L. & Croteau R. (1999) Monoterpene biosynthesis. In Comprehensive 13, 495–499. Natural Products Chemistry: Isoprenoids Including Carotenoids and Steroids Tollsten L. & Bergström J. (1989) Variation and post-pollination changes in (ed. D.D. Cane), pp. 97–153. Elsevier, Amsterdam, The Netherlands. floral odors released by Platanthera bifolia (Orchidaceae). Nordic Journal Yoo H., Widhalm J.R., Qian Y., Maeda H., Cooper B.R., Jannasch A.S., . . . Of Botany 9, 359–362. Dudareva N. (2013) An alternative pathway contributes to phenylalanine Van Moerkercke A., Schauvinhold I., Pichersky E., Haring M.A. & biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotrans- Schuurink R.C. (2009) A plant thiolase involved in benzoic acid bio- ferase. Nature Communications 4, 2833. synthesis and volatile benzenoid production. The Plant Journal 60, 292– Zangerl A.R. & Berenbaum M.R. (2009) Effects of florivory on floral volatile 302. emissions and pollination success in the wild parsnip. Arthropod-Plant Inter- Van Moerkercke A., Haring M.A. & Schuurink R.C. (2011) The transcription actions 3, 181–191. factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 Zvi M.M., Shklarman E., Masci T., Kalev H., Debener T., Shafir S., . . . promoter at a MYB binding site specific for fragrant petunias. The Plant Vainstein A. (2012) PAP1 transcription factor enhances production of Journal 67, 917–928. phenylpropanoid and terpenoid scent compounds in rose flowers. New Van Moerkercke A., Galvan-Ampudia C.S., Verdonk J.C., Haring M.A. & Phytologist 195, 335–345. Schuurink R.C. (2012a) Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of Received 17 December 2013; received in revised form 11 February petals. Journal of Experimental Botany 63, 3157–3171. 2014; accepted for publication 18 February 2014