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 Floral volatiles have attracted humans’ attention since antiquity and have since then permeated many aspects of our lives. Indeed, they are heavily used in perfumes, cosmetics, flavourings and medicinal applications. However, their primary function is to mediate ecological interactions between flowers and a diverse array of visitors, including pollinators, florivores and pathogens. As such, they ultimately ensure the plants’ reproductive and evolutionary success. To date, over 1700 floral volatile organic compounds (VOCs) have been identified. Interestingly, they are derived from only a few biochemical networks, which include the terpenoid, phenylpropanoid/ benzenoid and fatty acid biosynthetic pathways. These pathways are intricately regulated by endogenous and external factors to enable spatially and temporally controlled emission of floral volatiles, thereby fine-tuning the ecological interactions facilitated by floral volatiles. In this review, we will focus on describing the biosynthetic pathways leading to floral VOCs, the regulation of floral volatile emission, as well as biological functions of emitted volatiles. Key-words: benzenoids; floral scent; florivory; phenylpropanoids; pollination; regulation; terpenoids; volatile organic compounds.

INTRODUCTION Plants are sessile organisms that need to constantly adapt to changing environments for their survival and reproduction. For this environmental adaptation, plants have evolved a wide array of specialized metabolites, also called plant secondary metabolites or plant natural products. To date, over 200 000 specialized metabolites have been described (Dixon & Strack 2003), out of which approximately 1% corresponds to floral volatile organic compounds (VOCs) identified in 90 different angio- and gymnosperm families (Knudsen et al. 2006). VOCs are lipophilic liquids with low molecular weight and high vapour pressure at ambient temperatures. Physical properties of these compounds allow them to freely cross cellular membranes and be released into the surrounding environment (Pichersky et al. 2006). Biosynthesis of VOCs occurs in all plant organs: roots, stems, leaves, fruits, seeds, as well as flowers, which were found to release the highest amounts and diversity of VOCs. In contrast to VOCs released from other plant organs, which are exclusively involved in plant defense, floral VOCs assume functions in Correspondence: N. Dudareva. E-mail: [email protected] 1936

both attraction of pollinators and defence against florivores and pathogens. Based on their biosynthetic origin, floral VOCs can be divided into three major classes: terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives (Fig. 1). In addition, sulphur- and nitrogen-containing compounds contribute to the attraction of pollinators to flowers by mimicking food or brood sources such as carrion or dung (Wiens 1978; Faegri & van der Pijl 1979; Jürgens et al. 2006). However, to date, little is known about the biosynthetic pathways leading to the formation of these compounds.

BIOSYNTHETIC PATHWAYS AND GENES INVOLVED IN THE FORMATION OF FLORAL VOLATILES Biosynthesis of terpenoid compounds Terpenoids are the largest class of floral volatiles and encompass 556 scent compounds, which are derived from two common interconvertible five-carbon (C5) precursors: isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP) (McGarvey & Croteau 1995). In plants, these C5 precursors are synthesized from two independent and compartmentally separated pathways, the mevalonic acid (MVA) and the methylerythritol phosphate (MEP) pathways, which contribute to terpenoid biosynthesis in a species- and/or organ-specific manner (Vranova et al. 2013). The MEP pathway operates in plastids (Hsieh et al. 2008) and is mainly responsible for the formation of volatile mono- (C10) and diterpenes (C20) (∼53 and ∼1% of total floral terpenoids, respectively) (Knudsen & Gershenzon 2006), whereas the MVA pathway is distributed among the cytosol, endoplasmic reticulum and peroxisomes (Simkin et al. 2011; Pulido et al. 2012), and gives rise to precursors for volatile sesquiterpenes (C15) (∼28% of total floral terpenoids). While being compartmentally separated, these isoprenoid biosynthetic pathways are connected via a metabolic ‘crosstalk’ mediated by yet unidentified transporter(s) (Bick & Lange 2003; Flügge & Gao 2005). Such connectivity of the pathways allows the MEP pathway, often with a higher carbon flux than the MVA route, to support biosynthesis of cytosolically formed terpenoids as was demonstrated in vegetative tissue (Laule et al. 2003; Ward et al. 2011), fruits (Gutensohn et al. 2013) and flowers (Laule et al. 2003; Dudareva et al. 2005; Ward et al. 2011). Indeed, the MEP pathway alone supports sesquiterpene biosynthesis in snapdragon flowers (Dudareva et al. 2005). Terpenoid research in flowers has predominantly focused on the isolation and characterization of terpene synthase © 2014 John Wiley & Sons Ltd

Floral volatiles

Figure 1. Major volatile classes emitted by flowers. Based on their biosynthetic origin, volatiles emitted by flowers can be grouped into one of the three major volatile classes: terpenoids, phenylpropanoids/benzenoid, and fatty acid derivatives. Each volatile class is represented by a few typical floral scent compounds.

(TPS) genes responsible for the final steps in terpenoid biosynthesis, while genes and cognate enzymes of the MVA and MEP pathways were mainly characterized from vegetative tissues (Cane 1999; Wise & Croteau 1999; Lange et al.

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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 catalysed by the GPP synthase (GPPS) (Fig. 2). This enzyme was found to be heterodimeric in Antirrhinum majus (snapdragon) and Clarkia breweri, both of which have a floral scent bouquet rich in monoterpene compounds (Tholl et al. 2004). Analyses of tissue-specific, developmental and rhythmic expression of the GPPS small subunit showed positive correlation between expression and monoterpene emission in snapdragon flowers (Tholl et al. 2004), whereas no such correlation 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

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Table 1. List of biosynthetic genes involved in final steps of floral volatile formation Volatile Monoterpenoids 1,8-Cineole Linalool

Myrcene

E-(β)-Ocimene Sesquiterpenoids α-Farnesene Germacrene D

Nerolidol Valencene Benzenoids/phenylpropanoids Benzaldehyde Benzylacetate Benzylbenzoate Eugenol Isoeugenol Isomethyleugenol Methylbenzoate

Methyleugenol Phenylacetaldehyde 2-Phenylethanol Phenylethylbenzoate Veratrole

Gene

Species

Reference

CitMTSL1 NsCIN CbLIS AmNES/LIS-1 TPS10 TPS14 Am1e20 AmOc15 AlstroTPS Am0e23 CitMTSL4

Citrus unshiu Nicotiana suaveolens Clarkia breweri Antirrhinum majus Arabidopsis thaliana A. thaliana A. majus A. majus Alstroemeria peruviana A. majus C. unshiu

Shimada et al. 2005 Roeder et al. 2007 Dudareva et al. 1996a Nagegowda et al. 2008 Ginglinger et al. 2013 Ginglinger et al. 2013 Dudareva et al. 2003 Dudareva et al. 2003 Aros et al. 2012 Dudareva et al. 2003 Shimada et al. 2005

AdAFS1 AdGDS1 VvGerD FC0592 AmNES/LIS-2 AcNES1 VvVal

Actinidia deliciosa A. deliciosa Vitis vinifera Rosa hybrida A. majus Actinidia chinensis V. vinifera

Nieuwenhuizen et al. 2009 Nieuwenhuizen et al. 2009 Lucker et al. 2004 Guterman et al. 2002 Nagegowda et al. 2008 Green et al. 2012 Lucker et al. 2004

AmBALDH CbBEAT PhBPBT PhEGS PhIGS CbIEMT AmBAMT PhBSMT1 PhBSMT2 CbIEMT PhPAAS RhPAAS RdPAR PhBPBT SlGOMT1

A. majus C. breweri P. hybrida P. hybrida P. hybrida C. breweri A. majus P. hybrida P. hybrida C. breweri P. hybrida R. hybrida R. damascena P. hybrida Silene latifolia

Long et al. 2009 Dudareva et al. 1998 Boatright et al. 2004 Koeduka et al. 2006 Koeduka et al. 2006 Wang et al. 1997 Murfitt et al. 2000 Negre et al. 2003 Negre et al. 2003 Wang et al. 1997 Kaminaga et al. 2006 Farhi et al. 2010 Chen et al. 2011b Boatright et al. 2004 Gupta et al. 2012

small subunit is responsible for the regulation of GPP and subsequently monoterpene formation. Interestingly, a homodimeric GPPS with dual prenyltransferase activity (GPPS and FPP synthase activities) was reported in the orchid Phalaenopsis bellina and demonstrated to be linked to the emission of linalool and geraniol (Hsiao et al. 2008). FPP and GPP serve as substrates for TPSs and cyclases (Cane 1999; Wise & Croteau 1999), which in plants are responsible for the production of a vast variety of volatile terpenoid compounds (Fig. 2). TPSs are highly diversified throughout the plant kingdom and form a mid-size gene family (Bohlmann et al. 1998; Chen et al. 2011a), which is comprised of more than 100 genes identified in a variety of plant species, with one-third being isolated from flowers or fruits. Almost half of the known TPSs are capable of synthesizing multiple products from a single prenyl diphosphate precursor (Degenhardt et al. 2009). For instance, the floral volatile blend of Arabidopsis consists of 20 different sesquiterpenes, almost all of which are synthesized by only two sesquiterpene synthases, TPS11 and TPS21 (Tholl et al. 2005). The same is true for the flowers of kiwifruit (Actinidia

deliciosa), where almost all the sesquiterpenes released from flowers are the products of either germacrene D synthase1 (AdGDS1) or α-farnesene synthase1 (AdAFS1) (Nieuwenhuizen et al. 2009). In addition, some TPSs exhibit substrate promiscuity resulting in formation of different products. However, in the case of these TPSs their subcellular localization and the availability of a particular substrate determine the type of product formed (Tholl 2006; Nagegowda et al. 2008). In addition, the diversity of formed volatile terpenoids is not only dependent on TPSs, but is also increased by enzymes modifying TPS products by hydroxylation, dehydrogenation and acylation, which enhance their volatility and olfactory properties (Dudareva et al. 2004). To date, multiple flower-specific TPSs have been isolated and characterized (see Table 1). They were shown to be responsible for the formation of the monoterpenes linalool (C. breweri, A. majus and Arabidopsis thaliana) (Dudareva et al. 1996a; Nagegowda et al. 2008; Ginglinger et al. 2013), E-(β)-ocimene (A. majus and Citrus unshiu) (Dudareva et al. 2003; Shimada et al. 2005), myrcene (A. majus and Alstroemeria peruviana) (Dudareva et al. 2003; Aros et al.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1936–1949

Floral volatiles 2012) and 1,8-cineole (Nicotiana suaveolens and C. unshiu) (Shimada et al. 2005; Roeder et al. 2007), as well as of the sesquiterpenes nerolidol (A. majus and Actinidia chinensis) (Nagegowda et al. 2008; Green et al. 2012), α-farnesene (A. deliciosa) (Nieuwenhuizen et al. 2009), germacrene D (A. deliciosa, Rosa hybrid and Vitis vinifera) (Guterman et al. 2002; Lucker et al. 2004; Nieuwenhuizen et al. 2009) and valencene (V. vinifera) (Lucker et al. 2004). Besides mono- and sesquiterpenes, certain flowers also emit irregular terpenoids (C8 to C18). They constitute a minor class of floral terpenoids (∼7% of all floral terpenoids), which are formed via a three-step modification including a dioxygenase cleavage, enzymatic transformation and acidcatalysed conversion into volatile compounds (Winterhalter & Rouseff 2001). Interestingly, the dioxygenase cleavage step itself can already result in volatile products, such as α- and β-ionone, geranylacetone, and pseudoionone, as was found to be the case in petunia flowers (Simkin et al. 2004).

Biosynthesis of phenylpropanoid/ benzenoid compounds Phenylpropanoids and benzenoids represent the second largest class of plant VOCs (Knudsen et al. 2006) and are exclusively derived from the aromatic amino acid phenylalanine (Phe) (Fig. 3), which is synthesized via two alternative pathways (Maeda et al. 2010, 2011; Maeda & Dudareva 2012; Yoo et al. 2013). Depending on the structure of their carbon skeleton, this class is divided into three subclasses: phenylpropanoids (with a C6-C3 backbone), benzenoids (C6C1) and phenylpropanoid-related compounds (C6-C2). Phenylpropanoid-related compounds originate directly from Phe and constitute approximately 24% of all described phenylpropanoid/benzenoid compounds (Knudsen & Gershenzon 2006). So far, only genes and enzymes involved in the biosynthesis of phenylacetaldehyde and 2-phenylethanol have been isolated and characterized (Kaminaga et al. 2006; Sakai et al. 2007; Farhi et al. 2010; Chen et al. 2011b; Gutensohn et al. 2011; Hirata et al. 2012) (Fig. 3). In petunia petals, phenylacetaldehyde is produced via an unusual combined decarboxylation-amine oxidation reaction catalysed by phenylacetaldehyde synthase (Kaminaga et al. 2006). In roses, however, it is formed via two alternative routes: the first involves a phenylacetaldehyde synthase similar to the one described in petunia, while the second route employs Phe deamination by an aromatic amino acid aminotransferase followed by decarboxylation of the formed phenylpyruvate intermediate (Sakai et al. 2007; Farhi et al. 2010; Hirata et al. 2012). Further conversion of phenylacetaldehyde to 2-phenylethanol is catalysed by a phenylacetaldehyde reductase as was shown in roses (Sakai et al. 2007; Chen et al. 2011b). The first committed step in benzenoid (C6-C1) and phenylpropanoid (C6-C3) biosynthesis is catalysed by a well-characterized and widely distributed enzyme, Lphenylalanine ammonia-lyase (PAL), which deaminates Phe to trans-cinnamic acid (CA) and competes with phenylacetaldehyde synthase for Phe utilization (Fig. 3). Benzenoid

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formation from CA involves shortening of the propyl side chain by a C2 unit and was shown to proceed via a β-oxidative, a non-β-oxidative pathway or a combination of both (Boatright et al. 2004; Orlova et al. 2006) (Fig. 3). The β-oxidative pathway has only recently been fully elucidated in petunia flowers and appears to be analogous to fatty acid catabolism and is localized in peroxisomes. The pathway begins with an activation of CA to cinnamoyl-CoA, followed by hydration, oxidation and cleavage of the β-keto thioester with subsequent formation of benzoyl-CoA (Van Moerkercke et al. 2009; Klempien et al. 2012; Qualley et al. 2012). Benzaldehyde acts as the key intermediate in the alternative non-β-oxidative pathway and is oxidized to benzoic acid by a NAD+-dependent benzaldehyde dehydrogenase, which has been isolated and characterized from snapdragon flowers (Long et al. 2009). However, the enzymatic reactions leading to benzaldehyde formation remain unknown. Formation of floral phenylpropanoids (C6-C3), including (iso)eugenol and methyl(iso)eugenol, shares the initial biosynthetic steps with the lignin biosynthetic pathway up to the coniferyl alcohol stage. This monolignol precursor then undergoes two enzymatic reactions that eliminate the oxygen functionality at the C9 position. The first reaction involves acetylation by an acyltransferase from the benzylalcohol acetyl-, anthocyaninO-hydroxycinnamoyl-, hydroxycinnamoyl/benzoyl-CoA: anthranilate-N-hydroxycinnamoyl/benzoyl-, and deacetylvindoline acetyltransferases (BAHD) superfamily as was shown for the formation of coniferyl acetate from coniferyl alcohol in petunia petals (Dexter et al. 2007). Coniferyl acetate is then converted to the phenylpropanoids eugenol and isoeugenol by eugenol and isoeugenol synthases, respectively, which both belong to the pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase (PIP) family of NADPH-dependent reductases (Koeduka et al. 2006, 2008) (Fig. 3). In flowers, the diversity of phenylpropanoid/benzenoid compounds is further increased by modifications such as methylation, hydroxylation and acetylation of direct scent precursors. These modifications enhance the volatility or olfactory properties of scent compounds. Methylation reactions are catalysed by either O-methyltransferases or carboxyl methyltransferases. O-methyltransferases were shown to be responsible for the synthesis of a diverse array of benzenoids/ phenylpropanoids, including veratrole in Silene flowers (Gupta et al. 2012; Akhtar & Pichersky 2013), 3,5dimethoxytoluene and 1,3,5-trimethoxybenzene in roses (Lavid et al. 2002; Scalliet et al. 2002), and methyleugenol and isomethyleugenol in Clarkia (Wang & Pichersky 1998). Carboxyl methyltransferases, many of which belong to the s-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase, s-adenosyl-L-methionine: benzoic acid carboxyl methyltransferase, and theobromine synthase (SABATH) family (D’Auria et al. 2003), are involved in the biosynthesis of volatile esters like methylbenzoate in snapdragon and petunia flowers (Murfitt et al. 2000; Negre et al. 2003) and methylsalicylate in Clarkia and petunia (Ross et al. 1999;


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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 acyltransferases (D’Auria 2006) were shown to be responsible for the biosynthesis of acetylated scent compounds such as benzylacetate in Clarkia (Dudareva et al. 1998), benzoylbenzoate in Clarkia and petunia (D’Auria et al. 2002; Boatright et al. 2004; Orlova et al. 2006), and phenylethyl benzoate in petunia flowers (Boatright et al. 2004;Orlova et al. 2006).

Biosynthesis of volatile fatty acid derivatives Fatty acid derivatives constitute the third class of flower VOCs, which derive from the unsaturated C18 fatty acids, linolenic and linoleic. Biosynthesis of volatile fatty acid derivatives is initiated by a stereo-specific oxygenation of the octadecanoid precursors, catalysed by a lipoxygenase (LOX) and leads to formation of 9- and 13-hydroperoxy intermediates (Schaller


Floral volatiles 2001; Feussner & Wasternack 2002). These intermediates can enter two different branches of the LOX pathway, which in turn leads to the formation of volatile compounds. Allene oxide synthase (AOS) exclusively utilizes the 13-hydroperoxy intermediate as substrate, converting it to an unstable epoxide, which is then subjected to a cyclization followed by a reduction and a series of cyclization reactions to yield jasmonic acid (JA). In contrast to the AOS branch, hydroperoxide lyase can convert both types of hydroperoxide fatty acid derivatives into volatile C6 and C9 aldehydes. These saturated or unsaturated C6 and C9 aldehydes are often substrates for alcohol dehydrogenases giving rise to volatile alcohols, which can be further converted to their esters. These C6 and C9 aldehydes and alcohols are commonly referred to as green leaf volatiles, as they are usually synthesized in vegetative tissues. However, they are also important constituents in the floral volatile bouquet of several plant species such as carnation and wild snapdragon (Schade et al. 2001; Suchet et al. 2011). The orchids of the genus Ophrys produce an array of fatty acid-derived volatiles as well. Within their bouquet, alkenes are particularly important mediators in the interaction between orchids and their pollinators. Production of alkenes requires desaturation of fatty acids, a step that is likely mediated by acyl-acyl carrier protein (ACP) desaturases. Two isoforms of a stearoyl-acyl carrier protein (ACP) desaturase (SAD), namely SAD1 and SAD2, were identified in Ophrys sphegodes and O. exaltata. However, only expression of SAD2 was positively correlated with the formation of alkenes in flowers (Schlüter et al. 2011). OsSAD2 is a functional desaturase capable of producing 18:1Δ9 (ω-9) and 16:1Δ4 (ω-12) fatty acid intermediates from which 9-alkenes and 12-alkenes could be derived.

REGULATION OF FLORAL VOLATILE EMISSION Spatial, rhythmic and developmental regulation of floral scent emission Flowers have evolved many complex olfactory and visual guides for pollinator attraction. In order to maximize pollinator attraction, floral scent emission is often restricted to particular flower tissues and is developmentally and rhythmically regulated. Tissue-specific emission of floral VOCs is a characteristic feature of many species. In general, petals are the primary source of floral volatiles, although other tissues (stamens, pistils, sepals and nectaries) also contribute to the floral bouquet in certain plant species (Dobson et al. 1990, 1996; Bergström et al. 1995; Flamini et al. 2003; Dötterl & Jürgens 2005; Farré-Armengol et al. 2013). Emitted from petals, VOCs often enable long-distance attraction of pollinators, while VOCs produced in nectaries or pollen signal availability of food sources. Tissue specificity of scent emission is regulated at the level of scent biosynthetic gene expression and enzyme activity. Indeed, many of the scent biosynthetic genes isolated so far show a very specific expression profile, with the highest level found in the scent producing parts of the flower (see e.g. Dudareva et al. 1996a; Murfitt et al. 2000;

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Dudareva et al. 2003; Negre et al. 2003; Nagegowda et al. 2008; Rodriguez-Saona et al. 2011). Within scent-emitting tissues, formation of VOCs is often restricted to specific cell types or layers. In snapdragon flowers, for example, biosynthesis of the major volatile benzenoid compound methyl benzoate is restricted to the inner epidermal layer of the upper and lower petal lobes (Kolosova et al. 2001b). Similar cell-specific expression of scent biosynthetic genes was also reported for roses and C. breweri (Dudareva et al. 1996b; Bergougnoux et al. 2007). Rhythmicity of floral scent emission has been shown to occur in numerous species and often correlates with the activity of the respective pollinators (see e.g. Raguso et al. 2003; Dötterl et al. 2005; Effmert et al. 2005; Hoballah et al. 2005; Rodriguez-Saona et al. 2011). Rhythmic emission allows plants to conserve valuable carbon and energy during times of the day when their primary pollinators are inactive. Different modes of rhythmic scent release have been described so far. Diurnal rhythmicity in floral VOC emission was observed in plants pollinated during the day whereas nocturnally emitting plants are visited by pollinators foraging at night (Kolosova et al. 2001a; Waelti et al. 2008). Interestingly, the total amounts of emitted VOCs do not change over the day/night cycle in Dianthus inoxianus; however, the levels of compounds contributing to pollinator attraction vary according to visitor activity (Balao et al. 2011). Rhythmicity of scent emission is often transcriptionally regulated, similarly to its tissue specificity (see e.g. Kolosova et al. 2001a; Hendel-Rahmanim et al. 2007; Nagegowda et al. 2008; Nieuwenhuizen et al. 2009), although substrate availability for scent biosynthetic enzymes was shown to play a regulatory role in the emission of some compounds as well (Kolosova et al. 2001a). In addition to being spatially and rhythmically regulated, floral scent emission often changes over the lifespan of flowers. Usually, emission levels are highest when flowers are ready for pollination, that is, when anthers are dehisced, and decrease during senescence. Once pollinated, single flowers change or reduce the level of produced volatiles to prevent further visits potentially damaging the flower and to redirect visitors to the remaining unpollinated flowers (Schiestl & Ayasse 2001; Negre et al. 2003; Muhlemann et al. 2006; Rodriguez-Saona et al. 2011). Developmental regulation of scent emission occurs at several levels, including orchestrated expression of scent biosynthetic genes (Colquhoun et al. 2010), enzyme activities (see e.g. Pichersky et al. 1995; Dudareva et al. 2000; Shalit et al. 2003; Boatright et al. 2004; Nagegowda et al. 2008) and substrate availability (Dudareva et al. 2000).

Transcriptional network controlling volatile emission in flowers Orchestrated formation of volatiles from several independent pathways is not only a function of biochemical properties of biosynthetic enzymes, but also requires the involvement of transcription factors (TFs). Indeed, coordinated transcriptional regulation of entire scent biosynthetic networks has


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been recently shown (Colquhoun & Clark 2011; Muhlemann et al. 2012), implying that TFs control scent emission. Despite their importance, only a few TFs regulating the expression of scent biosynthetic genes have been identified to date. TFs controlling the flux through the phenylpropanoid/benzenoid network have recently been isolated from petunia flowers. ODORANT1 (ODO1), a R2R3type MYB TF, is exclusively expressed in petunia petal tissue and regulates the transcription of a major portion of the shikimate pathway as well as entry points into both the Phe (i.e. chorismate mutase) and phenylpropanoid (i.e. PAL) branchways (Verdonk et al. 2005). ODO1 was also found to activate the promoter of an ABC transporter of unknown function localized at the plasma membrane (Van Moerkercke et al. 2012a, 2012b). In petunia flowers, ODO1 is positively regulated by another R2R3-type MYB TF, EMISSION OF BENZENOIDS II (EOBII), which also activates the promoter of the biosynthetic gene isoeugenol synthase (Spitzer-Rimon et al. 2010; Colquhoun et al. 2011b; Van Moerkercke et al. 2011).The recently identified petunia EOBI was shown to be a flower-specific R2R3-type TF, which acts downstream of EOBII and upstream of ODO1 (Van Moerkercke et al. 2011; Spitzer-Rimon et al. 2012). Silencing of EOBI expression leads to down-regulation of numerous genes in the shikimate pathway (5-enolpyruvylshikimate3-phosphate synthase, 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase,chorismate synthase,chorismate mutase, arogenate dehydratase, and prephenate aminotransferase) as well as downstream scent-related genes (PAL, isoeugenol synthase, and benzoic acid/salicylic acid carboxyl methyltransferase) (Spitzer-Rimon et al. 2012). In contrast to ODO1, EOBI and EOBII, the MYB4 TF was found to be a repressor of only a single enzyme in the phenylpropanoid pathway, cinnamate-4-hydroxylase, thus controlling the flux towards phenylpropanoid volatile compounds in petunia flowers (Colquhoun et al. 2011a). While several TFs regulating the phenylpropanoid/ benzenoid network have been isolated and characterized, transcriptional regulation of the terpenoid pathways remains elusive. MYC2, a basic helix-loop-helix TF, was recently identified in Arabidopsis inflorescences and shown to activate the expression of two sesquiterpene synthase genes TPS11 and TPS21 via the gibberellic and JA signalling pathways (Hong et al. 2012). Despite the identification of several TFs for individual pathways, master regulators, which orchestrate formation of diverse volatile blends and act upstream of multiple metabolic pathways, are yet to be discovered. Recently, up-regulation of terpenoid and phenylpropanoid pathways was achieved by overexpression of the production of anthocyanin Pigment1 TF in roses (Zvi et al. 2012). However, it remains unknown whether promoters of genes involved in terpenoid formation are the natural targets for this TF.

Changes in floral volatile emission upon diverse biotic interactions In addition to internal regulatory mechanisms, several external factors are known to influence composition, quantity and

timing of volatile emission. These factors include pollination and interactions with floral antagonists. Successful pollination leads to a decrease or alterations in floral VOC emission in a variety of plant species (Tollsten & Bergström 1989; Tollsten 1993; Schiestl & Ayasse 2001; Negre et al. 2003; Theis & Raguso 2005; Muhlemann et al. 2006; Rodriguez-Saona et al. 2011). Besides reducing carbon loss caused by emission, post-pollination changes in VOCs redirect pollinators to yet unpollinated flowers (Schiestl & Ayasse 2001) or prevent additional visitation of pollinated flowers by flower antagonists (Muhlemann et al. 2006). Post-pollination decrease in emission was shown to occur within 24–96 h of pollinator visitation (depending on the plant species) and appears to be triggered once the pollen tubes reach the ovary (Negre et al. 2003). Upon fertilization, different molecular mechanisms were found to trigger the decrease in scent emission. In petunia flowers, reduced methylbenzoate emission after pollination is the result of transcriptional down-regulation of the cognate gene in an ethylene-dependent manner (Negre et al. 2003). In snapdragon flowers, however, the post-pollination decrease in methylbenzoate emission largely depends on reduced S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and substrate availability (Negre et al. 2003). Altered volatile emission was also reported in the context of above- and belowground plant–fungus interactions. Infection of Silene latifolia flowers by the anther smut fungus Microbotryum violaceum results in decreased total scent emission and discrimination against infected flowers by the pollinator (Dötterl et al. 2009). The intensity and chemodiversity of floral scent emission also decrease as a function of colonization by arbuscular mycorrhizal fungi in Polemonium viscosum, suggesting that plant–microbe interactions occurring outside of floral tissues can also modulate flower traits providing an additional layer of external regulatory mechanism (Becklin et al. 2011). Herbivore-induced plant volatiles (HIPVs) have been extensively studied in vegetative tissues. Signal perception and transduction mechanisms, as well as kinetics of HIPV release, are well characterized in these tissues (Dicke & Baldwin 2010). However, only few studies have linked florivory with induction of defence VOCs in flowers. One of the few known examples of florivore-induced volatile emission is represented by wild parsnip flowers, which emit higher amounts of octyl esters upon infestation with the parsnip webworm (Zangerl & Berenbaum 2009). Likewise, Helicoverpa zea larvae feeding on cotton flower buds induce emission of terpenes and fatty acid derivatives (Rose & Tumlinson 2004).

FLORAL VOLATILES AS MEDIATORS IN BIOTIC INTERACTIONS Floral VOCs possess multifaceted functions significantly contributing to attraction of pollinators and serving as defence compounds against pathogens and florivores. Although it was proposed that floral VOCs first served in protecting reproductive structures against antagonists and only later acquired


Floral volatiles pollinator-attracting capacities (Pellmyr & Thien 1986), their latter function has been studied the most. Pollinator attraction is mostly mediated by benzenoids whereas defence functions are predominantly assured by terpenoid and benzenoid VOCs (Schiestl 2010).

Floral volatiles in pollinator attraction For countless cross-pollinating plant species, mating involves the movement of pollen from one individual to another. In many cases, animals such as insects, birds and mammals significantly contribute to pollination by serving as vectors in pollen transfer. Flowers employ a diverse palette of signals to mediate attraction of pollinators to flowers for ensuring successful reproduction. For pollinators, this multisensory (visual, olfactory, thermal, electromagnetic) input is essential to locate food and breeding sites. Over the last decade, mounting evidence was accumulated for the role of floral volatiles in plant–pollinator communication. It has been shown that the information conveyed by floral volatiles depends on amount, composition and context of their emission, and elicits distinct behavioural responses in the respective pollinators. Long-distance emission of volatiles mainly contributes to guiding pollinators to flowers and is especially important for night-emitting plants where production of volatiles has to be of high intensity to overcome decreased conspicuousness of flowers under low illumination. In fact, the moth-pollinated Petunia axillaris and S. latifolia emit higher amounts of volatiles than day-emitting bee-pollinated plants within the same genus, like Petunia integrifolia and Silene dioica (Ando et al. 2001; Waelti et al. 2008). In contrast, volatiles emitted over short distances trigger landing, feeding and reproductive behaviour. Exposure even to a single floral volatile of the host plant S. latifolia elicited landing and feeding behaviour in Hadena bicruris moths (Dötterl et al. 2006) while subjection of nocturnal hawkmoth Manduca sexta to an olfactory stimulus induced proboscis extension (Goyret et al. 2007). Not only volatiles emitted from flower petals but also pollen odour can contribute to pollinator foraging. Indeed, pollen odour artificially added to antherless Rosa rugosa flowers increased frequency of bumblebees’ pollen-collecting behaviour relative to odour- and antherless flowers (Dobson et al. 1999). Volatiles emitted from flowers not only advertise food availability but also mating and oviposition opportunities. Examples include certain orchids that employ flower scent to imitate pheromone blends of female pollinators, thereby triggering copulation attempts of male pollinators with flowers (Schiestl et al. 1999). The dioecious species S. latifolia represents another example where floral volatiles provide oviposition cues for the females of the nursery moth pollinator H. bicruris, which lay eggs while pollinating the flowers (Brantjes 1976; Waelti et al. 2009). To mimic oviposition sites, a number of species within the five plant families (Araceae, Rafflesiaceae, Annonaceae, Apocynaceae and Orchidaceae) emit sulphur-containing volatile compounds to attract necrophagous, saprophagous and caprophagous insects. Interestingly, these volatiles, typically released by decompos-

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ing plant and animal organic matter, were found to arise independently via convergent evolution (Jürgens et al. 2013). These emitted volatiles also represent a characteristic feature of plants pollinated by necrophagous flies and beetles, thus showing a clear link between certain pollinator guilds and specific floral scent chemistries. Attempts to correlate floral volatile profiles with pollination syndromes have succeeded for certain plant–pollinator interactions; however, clear-cut predictions of pollinator guilds based on floral scent chemistry still remain unattainable. It was nevertheless established that plant species relying on moths for pollination predominantly release benzenoid, terpenoid and nitrogen-containing compounds (Knudsen & Tollsten 1993; Dobson 2006), and that bat-pollinated species mostly emit sulphur-containing volatiles (von Helversen et al. 2000). While involvement of volatiles in the attraction of hummingbirds remains debated, certain ornithophilous plants were found to emit minute amounts of terpenoids and fatty acid derivatives (Knudsen et al. 2004). Furthermore, hummingbirds were shown to display different behaviours (attraction or aversion) depending on the volatiles contained in the nectar of artificial flowers (Kessler & Baldwin 2007; Kessler et al. 2008).

Floral volatiles in flower defence Flowers are generally deficient in physical barriers such as a highly lignified cell wall and/or an impermeable cuticle, making them highly susceptible to pathogens and florivores. Moreover, they typically carry higher densities of microorganisms than other aerial plant surfaces due to their high moisture and nutrient content (Johnson & Stockwell 1998). Thus, flowers employ volatiles as an alternative mechanism that prevents damage to their reproductive structures. Many VOCs were shown to exhibit antimicrobial and antifungal activities in vitro (Bakkali et al. 2008) or inferred to have these antimicrobial activities due to tissue-specific expression patterns (e.g. in nectaries and/or stigmas) of their biosynthetic genes (Dudareva et al. 1996a; Chen et al. 2003). However, only few VOCs have been investigated for their role in defence against pathogens. (E)-β-caryophyllene emitted from stigmas of Arabidopsis flowers was shown to limit bacterial growth. Indeed, Arabidopsis plants lacking (E)-β-caryophyllene emission displayed denser bacterial populations on their stigmas and reduced seed weight compared with wild-type plants, indicating that (E)-βcaryophyllene acts in the defence against pathogenic bacteria and is important for plant fitness (Huang et al. 2012). VOCs emitted by Saponaria officinalis petals were shown to inhibit bacterial growth and suggested to control diversity of bacterial communities in petals (Junker et al. 2011b). Besides pathogens, florivores also cause substantial damage to reproductive tissues. Florivores are detrimental to plant fitness as they feed on reproductive structures, often displace potential pollinators and alter flower morphology (McCall 2008; Sõber et al. 2010). Similar to green tissue volatiles, floral volatiles are capable of deterring insects that are detrimental to pollinator visitation and/or feeding on


1944 J. K. Muhlemann et al. flower tissues. Ants are very inefficient pollinators (due to their morphology and mobility), often exhibit aggressive behaviour against pollinators and occasionally feed on floral structures. It was shown that many common European ants are deterred by volatiles emitted from temperate flowers (Willmer et al. 2009) and that inhibition of terpene biosynthesis leads to loss of ant-repellent properties (Junker et al. 2011a). The facultative florivore Metrioptera bicolor displays a strong aversion to linalool and (E)-β-caryophyllene, both of which are widespread terpenoid constituents of scent bouquets emitted by flowering species (Junker et al. 2010). These examples strongly suggest involvement of floral volatiles in deterrence against undesirable floral visitors.

Balance between defensive and attractive functions of floral volatiles Pollinators and florivores navigate the same visual and olfactory landscape to locate host plants. To avoid visitation by florivores while advertising their flowers to pollinators, plants have to balance attracting and deterring functions of floral volatiles. Unbalanced production of volatiles involved in different functions may result in negative impacts on plant fitness, as was shown in the cucurbit Cucurbita pepo where enhancement of floral fragrance led to higher attraction of florivores significantly affecting reproductive success (Theis & Adler 2012). Several strategies employed by flowers to optimize these functions and therefore maximize reproductive success have been described so far. In Cirsium arvense, some floral volatiles are responsible for attraction of both pollinators and florivores (Theis 2006). In this species, timing of emission is fine-tuned to increase likelihood of pollinator rather than florivore visitation. Indeed, developmental and diel timing of C. arvense volatile emission was found to be positively correlated with the flowers’ reproductive maturity and peak activity of pollinators, respectively, while negative correlation between diel emission and activity of florivores was observed (Theis et al. 2007). Similar to C. arvense, VOCs of petunia flowers attract both pollinators and florivores. Within the petunia VOC profile, some compounds specifically control infestation rate by florivores (i.e. isoeugenol and benzylbenzoate) whereas methylbenzoate, for example, is involved in pollinator attraction. Indeed, utilization of various petunia transgenic lines with down-regulation of different floral scent biosynthetic genes allowed elucidation of the distinct roles of individual volatile compounds in attraction of mutualists and deterrence of antagonists (Kessler et al. 2013). A similarly complex picture was found in the cucurbit Cucurbita moschata, where some compounds are attracting both pollinators and florivores, while other compounds are only mediating one type of interaction (Andrews et al. 2007). As a consequence of interactions with both mutualists and antagonists, different types of selection may act on the different volatile compounds. Indeed, compounds involved in attraction of both mutualists and antagonists will more likely be under balancing selection, while compounds involved in interactions solely with one type of flower visitor

are under directional selection pressure. This diversity of selection mechanisms is predicted to lead to the evolution of very complex floral volatile profiles. Nursery pollination systems represent a special case where insects simultaneously pollinate the flowers and use them as breeding sites. Flowers in these systems have evolved adaptive mechanisms to reduce damage caused by developing larvae that feed on the plant’s reproductive tissues (usually seeds) (Dufaÿ & Anstett 2003). In general, damage to seeds is predicted to be proportional to the number of visiting pollinators. Therefore, fast cessation of floral advertisement after successful pollination is essential to avoid further damage to the plant’s reproductive success. Indeed, a postpollination decrease specifically in pollinator-attracting volatiles was observed in Silene, thereby providing a mechanism to prevent further loss of seeds (Muhlemann et al. 2006).

EVOLUTION OF FLORAL SCENT The evolution of angiosperms has resulted in an immense diversity of flower traits such as shape, size, colour and scent. To date, more than 1700 floral volatile compounds have been described in over 900 flowering plant species (Knudsen et al. 2006). Interestingly, the quality and quantity of emitted volatiles are species specific and vary among different populations of a given species (Raguso 2008). While much effort has so far been invested in describing scent composition in various flowering species, the mechanisms driving the evolution and diversification of floral scent remain underexplored. Evolution of floral scent is potentially shaped by two factors that mutually influence each other: (1) genomic changes allowing catalytic expansion and differential regulation of the enzymatic machinery underlying floral scent formation and (2) ecological constraints such as pollinator-mediated selection. To date, only a few studies have examined the genetic basis for odour differences between closely related flowering species. P. axillaris and P. exserta represent a good example of a closely related species with distinct pollination syndromes. While P. axillaris flowers are colourless, emit benzenoid compounds and are moth pollinated, P. exserta flowers are red, devoid of scent and attract hummingbirds. Analysis of the genetic basis for differences in scent profiles between these two species revealed that only two quantitative trait loci are responsible for the distinct scent phenotypes (Klahre et al. 2011). One of these loci maps to the MYB TF ODO1, which controls flux through the shikimate pathway and hence the amount of precursors available for benzenoid biosynthesis (Verdonk et al. 2005), while the genetic identity of the second locus is presently unknown. C. breweri and C. concinna provide another example of two closely related scented and non-scented species, which rely on different pollinators. Although non-scented C. concinna contains genes responsible for formation of VOCs (i.e. linalool, (iso)methyleugenol and benzylacetate), transcriptional and/or post-transcriptional regulatory mechanisms lead to lack of their expression in flowers and elimination of floral


Floral volatiles scent (Raguso & Pichersky 1995; Dudareva et al. 1996a; Cseke et al. 1998; Nam et al. 1999). Differences in transcriptional levels were also found in the flowers of the orchid genus Ophrys, which emit a blend of fatty acid-derived volatiles (Schiestl et al. 1997, 1999; Schiestl & Ayasse 2002).Within this blend, alkenes are the key components for the attraction of pollinators to flowers and each Ophrys species emits a unique alkene profile, resulting in reproductive isolation between species through attraction of distinct and highly specific pollinators (Schiestl & Ayasse 2002). O. sphegodes mostly emits 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 action of a SAD. Differences in transcript levels, as well as changes in the tertiary structure of SAD2 between O. sphegodes and O. exaltata, were proposed as possible mechanisms underlying the distinct alkene profiles and reproductive isolation (Schlüter et al. 2011). While large intra- and interspecific variations in alkene profiles were detected within the Ophrys genus, only limited genetic variation among species and populations was observed with microsatellite markers (Mant et al. 2005). These findings suggest that divergent pollinator-mediated selection rather than genetic drift explains strong differences in volatile profiles. Taken together, the above examples demonstrate that small genetic variations can have large effects on floral scent chemistry and interactions with pollinators. Besides genetic polymorphisms, selection by pollinators is also capable of driving floral diversification and specialization. Pollinator-mediated selection is constrained by the pollinator’s pre-existing preferences and sensory abilities, and can occur only when the traits under selection are heritable and exhibit variation (Schiestl 2010; Schiestl & Dötterl 2012). Selection on floral scent by pollinators has been described in Penstemon digitalis, which displays marked inter-population 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 (Parachnowitsch et al. 2012). Several studies in other plant species have also uncovered volatile compounds that are important for plant–pollinator interaction and thus serve as potential targets for pollinator-mediated selection (see e.g. Dötterl et al. 2006; Shuttleworth & Johnson 2010; Schiestl et al. 2011). Interestingly, changes in floral scent through external manipulation (genetic or chemical) or intrinsic processes (mutations or hybridization) were shown to drive pollinator niche shifts, which are a proximate cause of floral and reproductive isolation. Indeed, Shuttleworth & Johnson (2010) have demonstrated that the presence/absence of sulphur compounds within the bouquets of four Eucomis species determines whether wasps or flies pollinate individual species. Furthermore, creation of more similar floral volatile profiles between S. dioica and S. latifolia by artificial manipulation of a single compound resulted in higher interspecific pollen transfer (Waelti et al. 2008). Thus, the aforementioned studies show that floral scent is an important factor in defining and maintaining species boundaries. They also demon-

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strate that loss or generation of reproductive isolation can occur through relatively simple changes in floral scent profiles. Evolution of reproductive isolation through changes in floral scent chemistry can occur within very short time frames, as was illustrated in Ophrys arachnitiformis x O. lupercalis hybrids (Vereecken et al. 2010). Indeed, generation of novel floral volatile profiles in these hybrids led to attraction of a pollinator species that does not pollinate any of the parent species (Vereecken et al. 2010), thereby leading to rapid floral isolation.

CONCLUSIONS AND FUTURE PERSPECTIVES Over the last two decades, the field of floral volatile research has acquired an ever-increasing amount of knowledge on the functions and biosynthesis of floral scent. Numerous floral volatile compounds have been identified to date from nearly 1000 plant species and their importance in mediating ecological interactions with floral mutualists and antagonists has been highlighted in many plant species. Recent advances in the isolation and characterization of genes and enzymes involved in different scent biosynthetic pathways, as well as in the elucidation of regulatory networks controlling these pathways, have also enhanced our understanding of how floral volatile compounds are synthesized. Despite recent progress in floral volatile research, many aspects of floral volatile function and biosynthesis remain largely unknown. In particular, we still do not know how the majority of floral VOCs are synthesized, how their orchestrated emission is regulated, and the specific roles of floral VOCs in large plantmutualist/antagonist interaction webs. We therefore anticipate that future research efforts will focus on providing insights into these specific aspects of floral scent biology and allow for development of defensive strategies as well as enhancement of yields in insect-pollinated plants.

ACKNOWLEDGMENTS This work was supported by grants from the National Science Foundation (MCB-0919987) and USDA-NIFA (2011-6701330126) to N.D.

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Received 17 December 2013; received in revised form 11 February 2014; accepted for publication 18 February 2014
