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Functional Characterization of Enzymes Forming VolatileEsters from Strawberry and Banana[w]

Jules Beekwilder*, Mayte Alvarez-Huerta1, Evert Neef, Francel W.A. Verstappen, Harro J. Bouwmeester,and Asaph Aharoni

Plant Research International, 6700 AA, Wageningen, The Netherlands

Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcoholacyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated fromfruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene fromthe cultivated strawberry (Fragaria 3 ananassa). The potential role of these enzymes in fruit flavor formation was assessed. Tothis end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol andacyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the variousmembers of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequencesimilarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation ofesters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significantextent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess thepotential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants over-expressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detectedin transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic linesresulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is animportant parameter to consider when engineering volatile ester formation in plants.

Volatile esters are produced by virtually all soft fruitspecies during ripening. They play a dual role in theripe fruit, serving both as ‘‘biological bribes’’ for theattraction of animals and as protectants against patho-gens. In some fruits, like apple (Malus domestica), pear(Pyrus communis), and banana (Musa sapientum), estersare the major components in their characteristicaroma. In other fruits, like strawberry (Fragaria 3ananassa), they contribute as notes to the blend ofvolatiles that constitute the aroma. Often, a single fruitemits a large spectrum of esters; for instance, morethan 100 different esters have been detected in ripestrawberry fruit (Zabetakis and Holden, 1997). Mostvolatile esters have flavor characteristics described asfruity (Burdock, 2002). However, they are not onlyproduced and emitted by fruit but also are part offloral scents (Dudareva et al., 1998b). They are veryoften emitted by vegetative parts of plants, eitherconstitutively or in response to stress or insect in-festation (Mattiacci et al., 2001). Esters therefore playa major role in the interaction between plants and theirenvironment. Formation of volatile esters is notrestricted to the plant kingdom and is also commonin microorganisms such as yeast and fungi. For exam-ple, isoamyl acetate and ethyl acetate are produced

during beer fermentation and are known to affectflavor quality (Verstrepen et al., 2003).

Alcohol acyltransferase (AAT) enzymes catalyze thelast step in ester formation by transacylation from anacyl-CoA to an alcohol. Combinations between differ-ent alcohols and acyl-CoAs will result in the formationof a range of esters in different fruit species. The mostlikely precursors for the esters are lipids and aminoacids. Their metabolism during ripening will thereforeplay an important role in determining both the levelsand type of esters formed. For example, in strawberry,the amino acid Ala has been implicated in the forma-tion of ethyl esters during ripening (Perez et al., 1992).However, levels of amino acid precursors could notexplain in all cases the formation of the correspondingester, and it has therefore been suggested that selec-tivity of enzymes preceding AATs in the biosyntheticpathway also plays a role in determining ester com-position (Wyllie and Fellman, 2000). In addition, fattyacids, generated by the oxidative degradation oflinoleic and linolenic acids, contribute to ester bio-synthesis in fruit. Degradation of fatty acids results inthe production of volatile aldehydes, which in turn areutilized by alcohol dehydrogenases to form alcohols.The latter are subsequently converted to hexyl andhexenyl esters (Perez et al., 1996; Shalit et al., 2001).Such a pathway has been demonstrated by Wang et al.(2001), who altered the levels of fatty acids in trans-genic tomato (Lycopersicon esculentum) fruit, leading todramatic changes in the aroma profile. Ripening re-lated processes, such as changes in cell wall composi-tion, might also contribute intermediates to esterbiogenesis. In tomato, evidence was provided that

1 Present address: Plant Physiology and Biochemistry Group,Institute of Plant Sciences, ETH Zurich, CH–8092 Zurich, Switzer-land.

* Corresponding author; e-mail [email protected]; fax 31–317–418094.

[w]The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.104.042580.

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activity of pectin methyl-esterase (which catalyzesdemetoxylation of pectins) regulates levels of metha-nol in the fruit (Frenkel et al., 1998). The accumulatingmethanol may then be utilized to generate methylesters in ripe fruit.

Due to their key role in ester biosynthesis, theactivity of AAT enzymes was the subject of severalearly investigations on extracts of various fruit species,including banana, strawberry, and melon (Cucumismelo; Wyllie and Fellman, 2000; Shalit et al., 2001; Oliaset al., 2002). In each of these studies, the AAT activitywas shown to be ripening induced, and the substratespecificity of the AATs toward tested substrates (bothto alcohols and acyl-CoAs) appeared to be broad. Inrecent years, fruit-expressed genes encoding enzymeswith AAT activity have been isolated and character-ized from strawberry and melon. Aharoni et al. (2000)utilized cDNA microarrays for gene expressionprofiling during strawberry fruit development andidentified the SAAT gene, which showed a stronginduction upon ripening. Moreover, the recombinantSAAT enzyme could catalyze the formation of esterstypically found in strawberry, using aliphatic,medium-chain alcohols (e.g. octanol) in combinationwith various chain length (up to C10 tested) acyl-CoAsas substrates. The activity of SAATwasmuch lower foraromatic substrates, and no activity could be detectedwith the monoterpene alcohol, linalool. Yahyaoui et al.(2002) reported on the molecular and biochemicalcharacterization of a ripening-induced and ethylene-regulated AAT gene (CM-AAT1) from ripe melon fruit.As in the case of SAAT, the recombinant CM-AAT1enzyme was capable of producing esters from a widerange of alcohols and acyl-CoAs. While CM-AAT1 andthe SAAT proteins share only 21% sequence identity,they show a similar preference toward alcohols.

Overall, the proteins encoded by both SAAT andCM-AAT1 showed low sequence identity to othergenes, but conserved motifs in their sequence couldassociate them to a plant superfamily of multifunc-tional acyltransferases, commonly referred to asBAHD (St-Pierre and De Luca, 2000). The first genesfrom this family that were biochemically characterizedwere the acetyl-CoA:benzylalcohol acetyltransferase(BEAT) from Clarkia breweri flowers, which forms theflower volatile benzyl acetate (Dudareva et al., 1998a)and the acetyl-CoA:deacetylvindoline 4-O-acetyltrans-ferase (DAT) from Catharanthus roseus (St-Pierre et al.,1998). The complete genome of Arabidopsis containsapproximately 60 genes annotated to belong to theBAHD family (D’Auria et al., 2002). In recent years,more members of this family have been cloned andcharacterized from a number of plant species. Some ofthese enzymes catalyze the formation of volatile esters,including benzyl alcohol benzoyl transferase (BEBT;D’Auria et al., 2002) from C. breweri flowers, makingbenzylbenzoate, acetyl-CoA:cis-3-hexen-1-ol acetyltransferase CHAT from Arabidopsis (D’Auria et al.,2002), which might be involved in the formation of cis-3-hexenyl acetate emitted upon injury, and RhAAT1,

suggested by Shalit et al. (2003) to be responsible forthe generation of geranyl and citronellyl acetate in roseflowers. In addition, many genes of the BAHD familythat are involved in the biosynthesis of nonvolatileesters have been isolated. They encode enzymes asso-ciated with the biosynthesis of phenylpropanoids(Fujiwara et al., 1998; Yonekura-Sakakibara et al.,2000), alkaloids (St-Pierre et al., 1998; Grothe et al.,2001), terpenoids (Walker and Croteau, 2000; Walkeret al., 2000), and shikimate pathway derivatives(Burhenne et al., 2003; Hoffmann et al., 2003).

In this study, we characterize the substrate prefer-ence of fruit-expressed members of the BAHD family.We cloned full-length cDNAs for enzymes from wildstrawberry (Fragaria vesca) and banana and comparedthem to SAAT. Recombinant expression in Escherichiacoli and assays on their ability to use different alcoholsubstrates provided evidence for a role of theseenzymes in the biosynthesis of esters that contributeto fruit flavor. Finally, we report the first attempt to usethe SAAT gene for metabolic engineering of plants,with the aim to alter the profile of emitted volatiles.

RESULTS

Further Characterization of the Cultivated Strawberry

SAAT Enzyme

The activity of recombinant SAAT enzyme wastested using a range of additional substrates that hadnot been tested by Aharoni et al. (2000). Substratepreference of the purified recombinant enzyme wastested in assays using 14C labeled acyl-CoAs as cosub-strates for a range of alcohol substrates. Substratepreference is used here as a measure to compare theactivity of an enzyme for different substrates at a fixedsubstrate concentration. In these tests, geraniol provedto be an excellent substrate. The activity with geraniolexceeded that with octanol (77% of activity withgeraniol), the best substrate in previous tests (Table I).Other alcohols including terpene alcohols (nerol,perilla alcohol, carveol, and farnesol) and aromaticalcohols (cinnamyl alcohol and eugenol) were alsotested. The activity of SAAT with nerol (54%) andperilla alcohol (45%) is significant, while the othersubstrates were only poorly used (Table I). The activityof SAAT with butyryl- and hexanoyl-CoA in combi-nation with a subset of alcohols was also studied(Table II). With the larger acyl-CoA cosubstrates, SAATagain preferred geraniol and C6 to C9 aliphatic alco-hols as substrates. However, relatively higher activitywas observed with butanol and hexanol in combina-tion with butyryl- and hexanoyl-CoA (81% and 76%,respectively), compared to acetyl-CoA (11%), whiledecanol is less preferred in combination with thehigher CoAs. This shows that SAATcan use a surpris-ing diversity of acyl-CoAs and alcohols, includingterpene alcohols, in addition to the aliphatic alcoholsthat are usually available in fruit.

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Table I. Substrate specificity of the SAAT, VAAT, and BanAAT recombinant enzymes toward differenttypes of alcohols

Comparison of esterification activity of each of the enzymes with different alcohols (20 mM) and 14Cacetyl-CoA (0.1 mM) as acyl donor.

Alcohola SAAT VAAT BanAAT Structureb

Methanol 5 6 2c,d 11 6 0.6 0.3 6 0.0

Ethanol 3 6 2d 11 6 0.9 0.1 6 0.0

1-Propanol 12 6 2e 18 6 0.1 n.t.f

2-Propanol 6 6 1e 1 6 0.1 n.t.

1-Butanol 11 6 1e 38 6 0.1 1 6 0.1

2-Butanol 15 6 0.1e 1 6 0.1 n.t.

Isoamylalcohol 17 6 1d 21 6 0.1 1 6 0.1

1-Hexanol 40 6 2e 39 6 0.1 n.t.

cis-2-hexen-1-ol 28 6 3d 39 6 0.0 n.t.

cis-3-hexen-1-ol 19 6 1d 45 6 0.0 n.t.

trans-2-hexen-1-ol 43 6 4d 100 6 0.7 31 6 0.4

trans-3-hexen-1-ol 6 6 0.4d 23 6 0.0 n.t.

1-Heptanol 70 6 25d 14 6 0.6 n.t.

1-Octanol 77 6 16e 6 6 0.0 44 6 0.3

1-Nonanol 66 6 1d 3 6 0.1 n.t.

1-Decanol 37 6 1d 1 6 0.1 n.t.

Furfuryl alcohol 3 6 0.4d 9 6 0.0 0.4 6 0.1

Benzylalcohol 3 6 0.2d 11 6 0.1 2 6 0.0

2-Phenyl-ethylalcohol 7 6 1d 12 6 0.4 1 6 0.0

Linalool 1 6 0.0e 1 6 0.9 n.t.

Geraniol 100 6 6 13 6 0.0 88 6 0.6

Nerol 54 6 2 3 6 0.1 46 6 0.3

Carveol 2 6 0.1 2 6 0.0 n.t.

Eugenol 7 6 0.0 7 6 0.7 n.t.

Farnesol 11 6 1.0 2 6 0.4 n.t.

Perilla alcohol 45 6 2 8 6 0.1 n.t.

2-Ethoxyethanol 2 6 0.3 4 6 0.1 n.t.

Cinnamyl alcohol 10 6 2 9 6 0.1 100 6 1

aThe added alcohol. bMolecular structure of the alcohol added. cActivity calculated as nano-moles product formed per hour per microgram protein. The highest activity for each enzyme was set at100%, and the numbers indicated signify the percentage of activity relative to the highest activity. ForSAAT, 100% was 3.0 nmol substrate h21 mg protein21; for VAAT 100% activity was 1.3 nmol substrate h21

mg protein21; for BanAAT 100% activity was 3.4 nmol substrate h21 mg protein21. dValues have beencalculated from data published previously (Aharoni et al., 2000). eValues have been calculated frompreviously published data and have been confirmed independently in the same experiment from whichthe novel data have originated. fn.t., Not tested.

Characterization of Enzymes Forming Fruit Esters

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Characterization of the Wild Strawberry (VAAT) and

Banana (BanAAT) Alcohol Acyl Transferases

Acyl transferase genes related to the SAAT genefrom strawberry were identified in a number of fruitspecies, as described in the supplemental data (avail-able at www.plantphysiol.org). From this collection,a gene from wild strawberry (termed VAAT) and frombanana (termed BanAAT) were selected for furtheranalysis. An alignment of the amino acid sequencesencoded by the cDNAs of SAAT, VAAT, and BanAAT isshown in Figure 1A.

The esters produced in wild and cultivated straw-berries are comparable (Pyysalo et al., 1979). There-fore, it was of interest to compare the SAATenzyme toits closest ortholog, VAAT, in more detail. First, se-quence comparison (Fig. 1A) reveals that the sequencesof both enzymes are closely related (86% identical).Diversity is concentrated in two regions in particular,around positions 60 and 430.

Also the substrate preference of the recombinantVAATand SAATenzymes were compared (Table I). Asreportedbefore (Aharoni et al., 2000), the SAATenzymehas a preference for C6 to C10 aliphatic alcohols. ForVAAT, the optimal size of aliphatic alcohols was clearlysmaller than for SAAT; only C6 alcohols (23%–100%)and n-butyl alcohol (38%) were good substrates, andmethyl and ethyl alcohols (11%), geraniol (13%), phe-nylethyl alcohol (12%), and benzyl alcohol (11%) wereused to limited extent. Longer aliphatic alcohols likeoctanol,which are favored by SAAT (77%),were hardlyused at all by VAAT (6%). So, although there is anoverlap between both enzymes for C6 alcohols, SAATand VAAT have shifted preferences.

The Km and Vmax values for octanol and acetyl-CoAwere determined for VAAT to compare these to the

published values of SAAT (Aharoni et al., 2000). TheKm value of VAAT for octanol, using 10 mM acetyl-CoAas a cosubstrate, is 0.6 mM (Vmax 5 89 nmol h21 mgprotein21), which is about 10-fold lower than that ofSAAT (Km 5 5.6 mM; Vmax 5 26 nmol h21 mg21). Thecatalytic efficiency of VAATwith octanol (expressed askcat/Km) is 1.0 3 103 M

21 s21, which is considerablyhigher than that of SAAT (32 M

21 s21). The Km value foracetyl-CoA (using 20 mM octanol as cosubstrate) isdramatically (200-fold) higher for VAAT (Km 5 2 mM;Vmax 5 350 nmol h21 mg21) than for SAAT (Km 50.01 mM; Vmax 5 45 nmol h21 mg21). The catalyticefficiency of SAATwith acetyl-CoA (3.1 3 104 M

21 s21)is about 20-fold higher than VAAT (1.2 3 103 M

21 s21).Recombinant expression in E. coli was also achieved

for BanAAT, and activity of the produced enzymecould be tested. In Table I, results with a limited set ofalcohol substrates in combination with acetyl-CoA arecompared to those obtained with SAAT and VAAT.The BanAAT enzyme showed an activity profile sim-ilar to SAAT, as it used geraniol, nerol, and the C6 andC8 alcohols quite efficiently. Cinnamyl alcohol was thebest substrate for BanAAT, while the SAAT and VAATenzymes were only very modestly active with thissubstrate.

Expression of SAAT in Transgenic Petunia

The activity of SAAT was also studied in plantausing petunia plants. Leaves of petunia do not emitany esters, while flowers emit exclusively esters orig-inating from benzyl-alcohol and methanol, in additionto nonester aromatic compounds (Verdonk et al.,2003). Plants were transformed with a cDNA encodingthe strawberry SAAT gene under control of the double35S-cauliflower mosaic virus promoter. All regener-ated plants were screened for the presence of the SAATgene by PCR. PCR-positive plants were transferred togreenhouse conditions for further examination. Trans-genic plants exhibited normal development, com-pared to nontransformed and empty-vector controlplants, which went through the same regenerationprocess.

Total RNA was extracted from young leaves of twonontransformed plants, three plants transformed withan empty binary vector, and six plants transformedwith SAAT. Expression of the SAAT transcript wasanalyzed by northern-blot analysis (Fig. 2). All ana-lyzed SAAT-transformed plants expressed the gene,but clear differences in expression level were ob-served. Plants S216A and S218A (low expressers)and S152A and S157A (high expressers) were selectedfor further analysis. T1-generation transgenic plantswere self-fertilized, and the T2 progeny was againselected for the presence of the SAAT gene by PCRscreening. From each line, three PCR-positive plantswere selected and compared to three nontransformedplants and three plants transformed with vectorpBinPLUS without an insert.

Table II. Substrate specificity of the SAAT recombinant enzymefor different types of alcohols in combination with acetyl-CoA,butyryl-CoA, and hexanoyl-CoA

Comparison of esterification activity of each of the enzymes withdifferent alcohols (20 mM) and 14C acetyl-CoA, butyryl-CoA, orhexanoyl-CoA (0.1 mM) as acyl donor.

Alcohol Acetyl-CoA Butyryl-CoA Hexanoyl-CoA

1-Butanol 11 6 2a 81 6 11 67 6 122-Butanol 15 6 1 75 6 1 50 6 51-Hexanol 40 6 2 73 6 23 65 6 4cis-2-hexenol 28 6 2 66 6 13 62 6 2cis-3-hexenol 19 6 5 43 6 5 n.t.b

trans-2-hexenol 43 6 4 81 6 15 100 6 121-Octanol 77 6 16 100 6 3 69 6 31-Nonanol 66 6 1 58 6 3 100 6 81-Decanol 37 6 1 6 6 2 15 6 6Benzyl alcohol 3 6 1 7 6 8 14 6 0.22-Phenylethyl alcohol 7 6 1 6 6 4 24 6 5Geraniol 100 6 6 89 6 5 75 6 3

aActivity calculated as nanomoles product formed per hour permicrogram protein. The highest activity for each enzyme was set at100%, and the numbers indicated signify the percentage of activityrelative to the highest activity. bn.t., Not tested.

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Figure 1. (Legend appears on following page.)





In Vitro Enzyme Activity of SAAT Produced by Petunia

Alcohol acyltransferase activity in leaves of theplants was analyzed in vitro, using acetyl-CoA andgeraniol as substrates. On three different days, ex-tracts from leaf material were assayed by addinggeraniol and acetyl-CoA. Formation of geranyl ace-tate was analyzed using gas chromatography-massspectrometry (GC-MS; Fig. 3). When the SAAT genewas not present (Control and pBinPLUS lines),hardly any geranyl acetate could be detected (Fig.3A), and plants with low SAAT-RNA levels (S216Aand S218A) produced no significant increase in theamount of geranyl acetate. High-expressing plants(S152A and S157A) produced 10 to 40 times moregeranyl acetate than low-expressers and controlplants (Fig. 3B, peak 3; Fig. 4A). It was concludedthat SAAT enzyme was expressed in active form inthese plants.

The Volatile Profile of Petunia Plants Producing SAAT

As a next step, the in vivo effect of the presence of theSAAT gene on volatile release of petunia was tested.The headspace volatilesweremeasured inflowers of allsix lines (S157A, S152A, S218, S216, pBinPLUS, andControl) for 2 d. No changes in the emission fromflowers of endogenous petunia esters (benzyl benzoateand methyl benzoate; Verdonk et al., 2003) were ob-served, nor were any esters or other volatile com-pounds that were novel to petunia detected (data notshown). Also in green parts, no changes in the emittedvolatiles could be observed (Fig. 5, A and B).

Activity of Petunia Expressed SAAT with ExternallySupplied Alcohols

To test if external substrates could be used by intactplant material, experiments were set up feeding

Figure 1. A, Comparison of the amino acid sequences of the VAAT, SAAT, and BanAAT enzymes. Amino acids shaded in blackrepresent identical matches; gray shaded boxes represent conservative changes. B, Nonrooted phylogenetic tree of the alcoholacyltransferase BAHD family members. Depicted are BanAAT (accession AX025506), VAAT (AX025504), and SAAT (AX025475;bold underlined); genes known to be expressed in fruit (bold, see supplemental data); and plant alcohol acyl transferases thathave been biochemically characterized. Fruit expressed genes are ApAAT (AX025508), ManAAT (AX025510), PAAT(AY534530), LAAT1 (AX025477), LAAT2 (AX025516), LAAT3 (AX025514), LAAT4 (AX25512), TomAAT (AY534531), andMAAT (AX025518). Transferases that have been biochemically characterized comprise anthocyan-5-aromatic acyltransferase(5AT; Q9ZWR8) from Gentiana triflora (Fujiwara et al., 1998), the hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6$-O-acyltransferase (3AT; BAA93475) from Perilla frutescens (Yonekura-Sakakibara et al., 2000), the DAT (AAC99311) fromCatharanthus roseus (St-Pierre et al., 1998), the 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT; Q9M6E2), and the2-alpha-hydroxytaxane 2-O-benzoyltransferase (TBT; Q9FPW3) from Taxus cuspidata (Walker and Croteau, 2000), the taxadien-5-alpha-ol O-acetyltransferase (TAT; Q9M6F0) from T. cuspidata (Walker et al., 2000), the 3#-N-debenzoyl-2#-deoxytaxolN-benzoyltransferase (DBTNBT; Q8LL69) from T. cuspidata (Walker et al., 2002), the BEAT (AAF04787) from Clarkia breweri(Dudareva et al., 1998a), the acetyl-CoA:cis-3-hexen-1-ol acetyl transferase (CHAT; AAN09797) fromArabidopsis (D’Auria et al.,2002), the salutaridinol 7-O-acetyltransferase (SalAAT; AAK73661) from Papaver somniferum (Grothe et al., 2001), the alcoholacyltransferase 1 (CM-AAT1; CAA94432) from Cucumis melo (Yahyaoui et al., 2002), the benzoyl-CoA:benzyl alcoholbenzyltransferase (BEBT; AAN09796) from C. breweri (D’Auria et al., 2002), the anthranilate N-benzoyltransferases fromDianthus caryophyllus (HCBT; CAB11466; Yang et al., 1997), andOrzya sativa (OsHCBT; AC114474; Burhenne et al., 2003); theagmatine coumaroyltransferase (HvACT; AAO73071) from Hordeum vulgare (Burhenne et al., 2003), and the geraniolacetyltransferase (RhAAT; BQ106456) from Rosa hybrida (Shalit et al., 2003).

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alcohol as a substrate. Initially, geraniol was used, butquantitative comparison of these experiments washampered by experimental problems. These problemsare likely related to the poor solubility of geraniol,which at 6 mM could only be temporarily suspended inwater. Plant parts that were in contact with the surfaceof the geraniol suspension showed severe necrosis,thus strongly affecting supply to the upper plant parts.When petunia leaves were fed with a solution ofisoamyl alcohol (which is soluble in water at 6 mM),no necrosis was observed and much more reproduc-ible results were obtained.Explants of petunia plants expressing the SAAT

enzyme produced abundant amounts of isoamyl ace-tate when fed with an isoamyl alcohol solution. Thisproduction depended on the presence of expressedSAAT enzyme (Peak 1, Fig. 5, C and D). In Figure 4B,

results on isoamyl acetate production of all six linesrecorded at three different days are presented. Highexpressers (S157A and S152A) reproducibly emittedabout 10-fold more isoamyl acetate than control leavesor low-expresser leaves. The SAATenzyme apparentlyfinds no suitable alcohol substrate in petunia unlessthis is fed to the plant.

When feeding alcohols to the petunia flowers, nochanges in the emission of endogenous petunia esters(benzyl benzoate and methyl benzoate; Verdonk et al.,2003) were observed (results not shown). Remarkably,in the leaf headspace, a few novel esters were detectedwhen isoamyl alcohol was fed independently from thepresence of the SAAT gene. The major esters (peaks 2and 3, Fig. 5C) were identified as 3-methyl butanyl2-methyl butanoate and 3-methyl butanyl 3-methylbutanoate (collectively referred to as isoamyl methyl-butyrate), while a minor peak (peak 4, Fig. 5C) wasidentified as isoamyl benzoate.

DISCUSSION

The Role of BAHD Family Members in FruitFlavor Formation

Esters are important for the flavor of a number offruits (Morton and MacLeod, 1990). Wild and culti-vated strawberries produce linear esters like ethylhexanoate, octyl acetate, and hexyl butyrate, whilebanana flavor is dominated by isoamyl acetate andbutyl acetate. Biosynthesis of esters in fruit is mediatedby enzymes from the BAHD family. Two fruit-expressed genes (SAAT from strawberry and CM-AAT1 from melon) from this family have been earlierimplicated in the biosynthesis of volatile esters(Aharoni et al., 2000; Yahyaoui et al., 2002). In thispaper, two other genes that are related to the SAATgene have been isolated from wild strawberry andbanana. Several lines of evidence suggest involvementof these genes in the formation of fruit flavor.

Figure 3. In vitro formation ofgeranyl acetate by leaf extracts ofwild-type petunia (A) and the SAATexpressing line S157A (B), whensupplied with geraniol and acetyl-CoA. Depicted are GC-MS chroma-tograms of m/z 69. The 100%detector response corresponds to2 3 106 ion counts. Major volatilesdetected are marked with numbers:Peak 1 is geraniol; peak 2 is nerol,a contaminant of the geraniol sam-ple; peak 3 is geranyl acetate.

Figure 2. Northern-blot analysis of RNA samples derived from petuniaplants. RNA was extracted from wild-type (Control 2 and Control 5)plants, plants transformed with empty vector pBinPLUS (pBIN24A,pBIN69A, and pBIN27A), and plants transformed with the pBIN-SAATconstruct (S152A, S216A, S249A, S157A, S136A, and S218A) andanalyzed for expression of the SAAT gene using amplified SAAT cDNAas a probe. In the last lane, an equal amount of strawberry RNA wasloaded. The bottom section represents a control hybridization witha petunia ubiquitin probe.





As a first criterion, the expression pattern of theVAAT and BanAAT genes was considered, basedon literature data. The VAAT gene (represented byclone 5.1.R2) was shown by RNA-gel blots of wild-strawberry fruit RNAs to be strongly induced inthe transitions between green and breaker fruit andbetween breaker and red fruit (Nam et al., 1999). Ina similar way, the BanAAT gene expression (repre-sented by cDNA clone UU130) was reported to be up-regulated in banana pulp after ripening was initiatedwith ethylene (Medina-Suarez et al., 1997).

As a second criterion, the sequence homology toknown acyltransferases was considered. As is clearfrom the presence of conserved sequences in the genes,they are part of the BAHD family (St-Pierre and De

Luca, 2000). Four groups within this family have beendefined by Hoffmann et al. (2003), based on sequencehomology. One group (A; Fig. 1B) comprises the Taxusacyltransferases involved in taxol biosynthesis, a sec-ond group (B) consists of the anthocyanin acyltrans-ferases, a third group (C) of genes encodes enzymeswith unrelated substrates, including SAAT, DAT, andBEAT, and a fourth group (D) comprises the hydroxy-cinnamoyltransferases. An additional fifth group,group E containing agamatine coumaroyltransferases,has been identified by Burhenne et al. (2003). Lastly,a group comprising the melon CM-AAT1 and wound-induced enzymes from C. breweri (BEBT) and Arabi-dopsis (CHAT) can be identified as group F. In Figure1B, the BanAAT and VAAT enzymes have been added

Figure 4. Activity of SAAT produced bypetunia. A, GC-MS analysis of the relativeamount of geranyl acetate formed in an invitro activity assay using leaf extracts of sixpetunia lines, acetyl-CoA, and geraniol.Assays were performed on three differentdays. Geranyl acetate was quantified byintegrating the peak surface area of m/z 69at 19.14 min. B, GC-MS analysis of therelative amount of isoamyl acetate emittedinto the headspace of stem explants withyoung leaves of six petunia lines, feedingon a 6.5 mM isoamyl alcohol solution, onthree different days. Quantity of emittedisoamyl acetate is expressed as microgramper gram fresh weight plant materialper 24 h.

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Figure 5. Total volatile emissionfrom petunia leaves. GC-MS chro-matograms (detector response;100% of 1.53 106 total ion counts)of head space volatiles released invivo by petunia leaves over a periodof 24 h. A, Leaves of wild-typepetunia leaves feeding on water. B,Leaves of line S157A on water. C,Leaves of wild-type petunia on anaqueous 6.5 mM isoamyl alcoholsolution. D, Leaves of line S157Aon an aqueous 6.5 mM isoamylalcohol solution. The major volatilecompounds detected are markedwith numbers: peak 1 is isoamylacetate; peak 2 is isoamyl 2-methylbutyrate; peak 3 is isoamyl 3-meth-ylbutyrate; and peak 4 is isoamylbenzoate.





to a phylogenetic tree including enzymes of the BAHDfamily with known activity and putative enzymesexpressed in fruit (see supplemental data). The VAATenzyme is very closely related to the SAATenzyme andfalls into group C. The BanAAT enzyme is exceptionaland does not match any of the sequence groups.

As a third criterion, the ability of fruit-expressedacyltransferases to produce the esters found in thefruits they were obtained from was tested. This wasdone by expressing the isolated cDNAs in E. coli, andtesting the ability of the recombinant and partiallypurified enzymes to form esters from a broad range ofalcohols in combination with acetyl-CoA. The SAATand VAAT enzymes are clearly able to produce anumber of the esters found in both wild and cultivatedstrawberries, such as hexyl acetate and octyl acetate.This further supports a role for the SAAT and VAATgenes in fruit flavor formation.

Also for the BanAAT enzyme, our data provideevidence for a role in flavor formation in ripeningbanana fruit, as recombinant BanAAT produced thecharacteristic banana volatiles such as isoamyl acetateand butyl acetate. However, these esters were madewith considerably lower efficiency than octyl acetate,cinnamoyl acetate, and geranyl acetate (Table I). An insitu analysis of AAT activity in banana fruit alsoshowed a preference for C6 alcohols over C3 and C4alcohols, as is the case for recombinant BanAAT, whilelonger alcohols like octanol were not tested (Wyllieand Fellman, 2000). This suggests that BanAAT corre-sponds to the major enzyme responsible for esterformation in banana fruit. Its low efficiency for syn-thesizing isoamyl acetate could explain the presence ofsignificant amounts of isoamyl alcohol (in addition toisoamyl acetate) in ripe banana volatiles (Shiota, 1993),which could be important for its volatile blend. Theexpression profile and substrate usage of the bananaand wild strawberry enzymes point to a role for theseenzymes in fruit flavor formation. However, definiteproof for this role has to come from reverse geneticsstrategies, which should show that fruits carryingknockout mutations or antisense-expressing con-structs of BAHD genes have clearly reduced emissionof esters.

A Comparison of Substrate Preferences of Related and

Unrelated Enzymes

A comparison of substrate preference can be madebetween the SAAT, VAAT, and BanAAT enzymes andrelated enzymes known from the literature. The closestrelative to SAAT for which activity measurementshave been published is the rose alcohol acyl trans-ferase (RhAAT; Shalit et al., 2003). The substratepreference of RhAAT is quite similar to that of SAAT.Both RhAATand SAAT prefer geraniol in combinationwith acetyl-CoA (Table II). For RhAAT, this preferencevery likely relates to the presence of geranyl acetate inrose flower scent. For SAAT, there is no obvious

relevance for this preference to the formation ofstrawberry fruit flavor, since geranyl acetate has notbeen reported to occur in ripe strawberries. VAAT iseven closer related to SAAT than RhAAT, but itsactivity is quite distinct from both enzymes. The VAATenzyme hardly accepts octanol, nonanol, or decanol assubstrates in combination with acetyl-CoA, althoughgeraniol is accepted. VAAT is much more active onshort alcohol substrates (C4 to C6, but also ethanol andmethanol), which are not preferred by SAAT, at least incombination with acetyl-CoA (Table I). These obser-vations are in agreement with the substrate prefer-ences reported for enzyme extracts of wild andcultivated strawberry fruits (Olias et al., 2002). Re-markably, the requirements of SAAT with respect toalcohols combined with butyryl- and hexanoyl-CoAare less stringent (Table II). Butanol, for instance, ishardly used with acetyl-CoA, while it is readilyaccepted with butyryl-CoA and hexanoyl-CoA. Thisis particularly relevant since butylbutyrate and butyl-hexanoate have been identified in strawberries(Zabetakis and Holden, 1997).

It is difficult to compare absolute kinetic parametersfor these enzymes in a way relevant for the in vivosituation. The catalytic efficiency of SAAT for octanolis much (30 times) lower than that of VAAT. On theother hand, the substrate preference data (Table I)indicate that SAAT turns over 2 nmol octanol per hourper mg protein, while VAAT turns over only 0.078 nmoloctanol per hour per mg protein, suggesting that SAATis 25-fold more efficient for this substrate. These seem-ingly contradictory observations can be explained bythe difference in affinity for the second substrate,acetyl-CoA, of both enzymes. Substrate preferencemeasurements were carried out at 0.1 mM acetyl-CoA,which ensures saturated substrate binding by SAAT(10 times higher than Km, which is 0.01 mM), but notby VAAT (20 times lower than Km, which is 2 mM).Obviously, this has strong impact on the observedsubstrate preferences. The in vivo concentration ofacetyl-CoA in strawberry fruit is not known, but forgreen leaves it has been reported to range from 0.05 to1.4 mM, depending on the number of chloroplastspresent (Bao et al., 2000). In view of the low amount ofplastids in strawberry fruit, the acetyl-CoA concentra-tion in this fruit is probably at the lower end of thisrange, which is why it was estimated that a concentra-tion of 0.1 mM acetyl-CoA in the substrate preferenceassay was appropriate.

The banana enzyme BanAAT is only distantly re-lated to the SAAT and VAAT, members of group C ofthe BAHD family (Fig. 1, A and B). However, BanAATshares most enzymatic properties with SAAT andRhAAT (Shalit et al., 2003) and prefers octanol andgeraniol. The VAAT preferences are more related tothose of CM-AAT1 (Yahyaoui et al., 2002), which doesnot accept nonanol, and BEAT and BEBT (Dudarevaet al., 1998a; D’Auria et al., 2002), which producebenzyl acetate. However, these enzymes belong togroup F, while VAAT belongs to group C. These cases

Beekwilder et al.




illustrate that predicting substrate usage based onsequence grouping remains difficult. A similar lackof relationship between primary structure and enzy-matic properties has recently been reported for thehydroxycinnamoyltranferases (Burhenne et al., 2003).Pichersky and Gang (2000) have suggested a special

form of convergent evolution in which new enzymeswith the same function evolve independently in sep-arate plant lineages from a shared pool of relatedenzymes with similar but not identical functions. Sucha situation seems to be the case with the fruit AATsthat are involved in the formation of volatiles. There isa strong overlap in substrate specificity of unrelatedmembers of the BAHD gene family (for instance SAATand BanAAT) and differences in specificity betweenclosely related members such as SAAT and VAAT.

Engineering of Volatile Esters in Plants

The engineering of volatile emission from plants hasbeen describedmainly for terpenoids. Lewinsohn et al.(2001) transformed a tomato variety, with a low en-dogenous level of linalool in the fruit, with C. brewerilinalool synthase under control of the fruit-specific E8promoter. This resulted in fruit with strongly elevatedlevels of S-linalool. The same gene was used to in-troduce the emission of linalool from carnation flowers(Lavy et al., 2002). Interestingly, when Lucker et al.(2001) attempted to realize linalool emission frompetunia, a plant that does not normally emit significantamounts of terpenes, hardly any change in volatileemission was observed. Overexpressing the C. brewerilinalool synthase resulted instead in accumulation oflinalool to significant amounts in the form of a non-volatile glucopyranoside conjugate. Thus, endogenousenzymes in petunia seem to sequester the volatilelinalool as a nonvolatile conjugate. Leaves of Arabi-dopsis plants constitutively expressing a linalool syn-thase from strawberry did produce linalool but also itsglycosylated and hydroxylated derivatives (Aharoniet al., 2003).The pathway eventually leading to ester formation

has been engineered by Speirs et al. (1998). By over-expressing alcohol dehydrogenase in tomato fruit,levels of hexanol and cis-3-hexenol were increased,at the expense of aldehydes. These fruits wererecognized by trained taste panels as having a moreintense ripe fruit flavor. By expressing SAAT in petu-nia, we aimed to engineer the emission of esters, whichcould convey a fruity aroma to the plant. However, asin the case of engineering linalool production inpetunia, engineering emission of volatile esters inplants appears not to be trivial. Both expression ofSAAT and the activity of its corresponding enzymecould be detected in the transgenic plants generated,and activity was found to be inherited in the T2generation. However, no new esters could be detectedin the headspace of transgenic lines, and no increase inthe ester benzylbenzoate, which is normally emitted

by wild-type petunia flowers, was observed. Feedingalcohols to petunia explants demonstrated that thebottle-neck for ester production was the availability ofalcohols. Clearly, the success of engineering of esterformation will also depend heavily on substrate avail-ability.

Our results indicate the presence of alcohol acyl-transferase activity also in nontransgenic petuniaplants. When fed with isoamyl alcohol, the wild-typeplant converted part of the alcohol into isoamylmethylbutyrate and isoamyl benzoate (Fig. 4C). Thisstrongly suggests that there is an alcohol acyltrans-ferase active in the petunia background. The fact thatbenzyl benzoate has been found in considerableamounts in wild-type petunia flowers (Verdonk et al.,2003) also suggests the presence of such an endoge-nous enzyme. Very low levels of isoamyl acetatewere found in wild-type plants when fed with isoamylalcohol (Fig. 4C), indicating that the putativeendogenous acyltransferase hardly uses acetyl-CoA,while it can use methylbutyryl-CoA and benzoyl-CoA.Probably both acetyl-CoA and benzoyl-CoA are avail-able in petunia, and while the endogenous enzymeuses only benzoyl-CoA, the ectopic SAATenzyme usesacetyl-CoA.

To engineer ester production into plants, it shouldbe considered that a supply of alcohol substrate isrequired, either from an endogenous source or byintroducing additional genes. The SAAT enzyme cantake many different alcohol substrates, which arederived from several pathways. Linear alcohols likeoctanol could be provided by stimulating fatty acidmetabolism, for instance by expressing lipoxygenasegenes, or by amino acid catabolism. Terpene alcoholslike geraniol could be provided by introducing a gera-niol synthase (Iijima et al., 2004). However, by engi-neering the alcohol supply, other side-products maybe induced. This is illustrated by our finding of pre-viously undetected isoamyl methylbutyrate and iso-amyl benzoate in the wild-type petunia headspacewhen feeding isoamyl alcohol (Fig. 5C). Engineering ofester formation in a background that already containsalcohols may avoid these problems. In fruits of tomatoand strawberry, for instance, alcohols are quite abun-dantly available (Zabetakis and Holden, 1997; Speirset al., 1998). Another possibility is to vary culturingconditions, for example by introducing stress, whichwill affect the concentration of alcohols (Mattiacciet al., 2001). Our findings parallel those describedby Schwab (2003), who reviewed several reports onbiochemical analysis of heterologously expressedenzymes where poor specificity was observed forenzymes involved in the production of secondarymetabolites during fruit ripening, (e.g. in flavor andcolor formation). It was concluded that metabolomediversity is also caused by low enzyme specificity anddirectly related to availability of suitable substrates.Future work will therefore have to take into accountthe entire metabolic pathway, rather than a single en-zyme, to engineer emission of novel volatiles in plants.





MATERIALS AND METHODS

Plant Material and Petunia Transformation

For cloning the full-length cDNAs, we used ripe fruit of wild strawberry

(Fragaria vesca L., PRI breeding line 92189) and pulp of ripe banana (Musa

sapientum). The petunia (Petunia hybrida [Vilm.]) variety W115 was used for

generating transgenic plants, which were grown at 18�C with 18/6 h light/

dark conditions in the greenhouse.

RNA Isolation and RNA-Gel Blots

RNA isolation from the various fruit tissues was performed according to

Asiph et al. (2000) and from leaves of petunia using the method of Verwoerd

et al. (1989). RNA-gel blots were generated (10 mg of either petunia leaf or ripe

strawberry fruit total RNA) and hybridized as described previously (Aharoni

et al., 2000). The SAAT gene probe (780-bp fragment) was generated by PCR

using the oligonucleotides AAP157 5#-TCCAATCCATGATGTCCTCC-3#, andAAP158 5#-GCTTGGAGTTCAAGTCAACG-3#. The blot was rehybridized

with a petunia cDNA probe showing homology to a gene encoding a ubiquitin

protein (identified in a petunia expressed sequence tag library described in

Aharoni et al., 2000) that was amplified by PCR from a pBlueScript vector

(Stratagene, La Jolla, CA) using the T3 and T7 oligonucleotides.

SAAT Overexpression Construct and

Plant Transformation

The entire SAAT coding region (1,359 bp) was amplified from pRSETB

(Aharoni et al., 2000) using oligonucleotides AAP165 (5#-CGGATCCGGA-

GAAAATTGAGGTCAG-3#) and AAP166 (5#-CGTCGACCATTGCACGAGC-

CACATAATC-3#). The amplified fragment was cleaved with BglII and SalI

restriction enzymes and inserted in a sense orientation into BamHI and SalI

restriction sites, located between a double 35S-cauliflower mosaic virus

promoter and a nopaline synthase terminator, in the pFLAP10 subcloning

vector (provided by A. Bovy, Plant Research International, Wageningen, The

Netherlands). From the pFLAP10 vector, the fragment was excised with PacI

and AscI restriction digestions and introduced to the pBinPLUS binary vector

(Van Engelen et al., 1995) containing the kanamycin resistance selection

marker (nptII). Petunia was transformed with the LBA4404 Agrobacterium

tumefaciens strain as described by Lucker et al. (2001). Fifteen lines rooting

vigorously on media containing kanamycin (75 mg/L) were transferred to the

greenhouse. Plants transformed with the empty pBinPLUS binary vector and

nontransformed wild-type ones were also regenerated in a similar way in

vitro on nonselective medium and utilized for the experiments.

Cloning Full-Length Genes, Sequencing, and

Phylogenetic Analysis

To obtain full-length cDNAs we used the SMART RACE cDNA amplifi-

cation kit (CLONTECH, Palo Alto, CA) according to the manufacturer

instructions with slight modifications to the annealing temperatures

(normally 5�C–10�C lower than recommended) or number of cycles (up to

35 cycles). PCR, restriction digests, plasmid DNA isolation, and gel

electrophoresis were performed using standard protocols. All fragments were

purified from the gel using the GFX purification kit (Amersham, Little

Chalfont, UK). Cloning of PCR fragments was either performed using the

PCR SCRIPT (Stratagene, La Jolla, CA) or pCR 4Blunt-TOPO (Invitrogen,

Carlsbad, CA) vectors (for blunt-end products generated when using Pfu

DNA polymerase) or to the pGEM-T Easy (Promega, Madison, WI) vector

(when A tailed PCR products were generated by the use of Taq DNA

polymerase). Sequencing was done using an ABI 310 capillary sequencer

according to the manufacturer instructions (ABI system, Perkin Elmer, Foster

City, CA). Sequence analysis was conducted using the DNASTAR (DNAS-

TAR, Madison, WI) and GeneDoc (http://www.cris.com/Ketchup/gene-

doc.shtml) programs. Multiple sequence alignments were performed using

ClustalW (at http://www.ebi.ac.uk/clustalw/), using standard parameters.

Cloning of the full-length wild strawberry cDNA homolog was performed

by PCR using oligonucleotide AAP165 (5#-CGGATCCGGAGAAAATT-

GAGGTCAG-3#) designed at the 5# end from the SAAT gene sequence

isolated previously from the cultivated strawberry by Aharoni et al. (2000) and

oligonucleotide AAP184 (5#-CGTCGACCGGATAACATACGTAGAC-3#) at

the 3# end. AAP184 was designed based on a partial cDNA clone (522 bp,

GenBank accession no. AJ001450.1), which showed high homology to SAAT

and had been described by Nam et al. (1999) from the wild strawberry.

To clone the full-length banana cDNA, RACE PCRwas performed (method

described above) in order to clone the 3# end using the AAP218 oligonucle-

otide (5#-TCATCTCCGTCCATACCATCG-3#) designed based on a partial

cDNA sequence (799 bp; GenBank accession no. Z93116) isolated previously

from banana pulp (Medina-Suarez et al., 1997). The products of the first PCR

were used for a nested PCR reaction using a second specific oligonucleotide

on the published sequence (AAP219; 5#-CACATCCATCACGTCAAGTCC-3#),and a 700-bp 3# end fragment was obtained. Based on the 3# end fragment

sequence, the AAP232 oligonucleotide was designed for a 5# RACE reaction

(5#-CCTCGAGGGATACTGACAAGTACTACAC-3#) in order to clone the

entire banana cDNA.

Expression and Partial Purification of theRecombinant Proteins

A modified expression vector (pRSET B; Invitrogen) described earlier

(Aharoni et al., 2000) was used for the expression of VAAT and BanAAT in

Escherichia coli cells (BL21 Gold DE3 strain; Stratagene). The oligonucleotides

used for cloning to pRSET B introduced restriction sites at both ends of the

fragments, which were compatible to the BamHI and SalI restriction sites in the

vector. The oligonucleotides AAP165 and AAP184 (see above) introduced

a BamHI and a SalI, respectively, to VAAT; AAP233 (5#-CGGATCCGAGCTT-

CGCTGTGACCAGAAC-3#) and AAP227 (5#-ACTTCACGTCCTCCAAGCT-

GAGTC-3#) introduced a BamHI and aXhoI, respectively, to BanAAT. To obtain

an in-frame N-terminus fusion of the recombinant proteins to the His-tag, the

A and T nucleotides of the ATG codon were removed, resulting in the

elimination of the starting Met and insertion of a Pro in the protein sequences.

Vectors pRSET-SAAT, pRSET-VAAT, and pRSET-BanAAT were trans-

formed in E. coli BL21 CodonPlus-RIL. Fresh overnight cultures were diluted

100-fold in 50 mL Luria-Bertani medium supplied with ampicillin (50 mg/mL)

and Glc (1%) and grown until A600 was 0.6. Cells were recovered by

centrifugation and resuspended in Luria-Bertani medium supplied with

ampicillin and isopropylthio-b-galactoside (1 mM). After overnight growth

at 16�C, cells were harvested by centrifugation and resuspended in 1 mL ice-

cold buffer R (50 mM Tris-HCl, pH 5 8, and 10 mM 2-mercaptoethanol). Ten

micrograms lysozyme was added, and cells were sonicated on ice for 5 times

10 s, with 5 s breaks and an amplitude of 14 using an MSE Soniprep 150

sonicator (MSE Scientific Instruments, Crawley, UK). Sonicated cells were

centrifuged at 4�C and 14,000g for 5 min. The supernatant was used for

purification of His-tagged proteins on a Ni-NTA spin column (Qiagen,

Valencia, CA) as prescribed by the manufacturer. Resulting eluates were

adjusted to contain 2 mM dithiothreitol, and were immediately used for

activity assays.

Substrate Preference Assays

Activity of recombinant proteins with different alcohol substrates was

assayed using 14C radiolabeled acetyl-CoA (Amersham) or 14C-butyryl-CoA

or 14C-hexanoyl-CoA (Campro, Veenendaal, The Netherlands). The reactions

were made in duplicate. In a total reaction volume of 100 mL, 2 mL acyl-CoA (5

mM), 0.2 mL 14C labeled acyl-CoA, 85.8 mL 50 mM Tris-HCl buffer, pH 8.3, and

10 mL purified enzyme were mixed slowly in a cold Eppendorf (Westbury,

NY) tube. The reactions were started by adding 2 mL alcohol, solved at 1 M in

hexane. After 30 min incubation at 30�C, the reactions were stopped by

cooling on ice for 15 min. The esters formed were extracted with 700 mL

hexane. The hexane phase was mixed with 4.5 mL scintillation cocktail

(Ultima Gold, Packard Bioscience, Groningen, The Netherlands) and analyzed

by scintillation counting. The enzymatic activity was determined as nmol

substrate turned over per hour per microgram protein. Km and Vmax determi-

nations for recombinant VAAT protein were performed exactly as described in

Aharoni et al. (2000) for SAAT. Kcat values were determined by dividing Vmax

values by the protein concentration of the purified enzyme in the reaction

mixture, taking into account the calculated molecular mass of about 50 kD.

Enzyme Activity Assays with Plant Material

One gram of leaf material was ground in a pestle and mortar and

immediately mixed into 1 mL buffer A containing 50 mM Tris-HCl, pH 8.0,

Beekwilder et al.




and 1 mM dithiothreitol. The material was centrifuged for 5 min at 13,000g,

and the supernatant was used for assays. The assay was performed in

a capped vial at 30�C and contained 50 mL supernatant, 300 mL buffer A, 25 mL

8 mM acetyl-CoA solution, and 25 mL 1.6 mM geraniol suspension. After

30 min, 400 mL saturated CaCl2 solution was added by injection through the

septum. Products were analyzed using solid-phase microextraction. Volatiles

released into the vial headspace (at 35�C with stirring) were subsequently

trapped for 10 min by exposing a 100-mm polydimethylsiloxane fiber

(Supelco, Bellefonte, PA) to the headspace. Desorption was performed for 1

min, and products were analyzed by GC-MS, as described by Verhoeven et al.

(1997).

Headspace Analysis

To analyze the headspace of plant parts, three flowers or three stem ex-

plants with a single node and two to three young leaves were cut from a single

plant and weighed and cut ends were placed together in a covered 10-mL

beaker covered with aluminum foil and containing 2 mL of water. For

headspace analysis (performed basically as described by Bouwmeester et al.,

1999), beakers with the detached plant parts were placed in closed glass cuvets

(500 mL) for 24 h at 21�C in a light regime of 9 h light, 8 h darkness, and 7 h

light. A vacuum pump was used to draw air through the glass cuvet at

approximately 100 mL/min, with the incoming air being purified through

a glass cartridge (140 3 4 mm) containing 150 mg of Tenax TA (20/35-mesh,

Alltech, Deerfield, IL). At the outlet, the volatiles emitted by the detached

leaves were trapped on a similar cartridge. Volatiles were collected during

24 h. Cartridges were eluted using 3 3 1 mL of redistilled pentane:diethyl

ether (4:1). Of the (nonconcentrated) samples, 2 mL was analyzed by GC-MS

using a gas chromatograph (5890 series II, Hewlett-Packard) equipped with a

30-m 3 0.25-mm i.d., 0.25-mm film thickness column (5MS, Hewlett-Packard,

Palo Alto, CA) and amass-selective detector (model 5972A, Hewlett-Packard).

The GC was programmed at an initial temperature of 45�C for 1 min, with a

ramp of 10�C/min up to 220�C and final time of 5 min. The injection port

(splitless mode), interface, and MS source temperatures were 250�C, 290�C,and 180�C, respectively, and the He inlet pressure was controlled with an

electronic pressure control unit to achieve a constant column flow of 1.0 mL/

min. The ionization potential was set at 70 eV, and scanning was performed

from 30 to 250 atomic mass units. Data were quantified by comparison to

injected standards.

Sequence data from this article have been deposited with the EMBL/

GenBank data libraries under accession numbers AJ001450.1, Z93116,

AX025506, AX025504, AX025475, AX025508, AX025510, AY534530,

AX025477, AX025516, AX025514, AX25512, AY534531, AX025518, Q9ZWR8,

BAA93475, AAC99311, Q9M6E2, Q9FPW3, Q9M6F0, Q8LL69, AAF04787,

AAN09797, AAK73661, CAA94432, AAN09796, CAB11466, AC114474,

AAO73071, BQ106456, pBIN24A, pBIN69A, pBIN27A, S152A, S216A,

S249A, S157A, S136A, and S218A.

ACKNOWLEDGMENTS

We acknowledge Jan Blaas and Harrie Verhoeven for assistance with

solid-phase microextraction analysis and Robert Hall for critical reading of

the manuscript.

Received March 12, 2004; returned for revision April 6, 2004; accepted April

16, 2004.

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