Characterization of an Acyltransferase Capable of Synthesizing Benzylbenzoate and Other Volatile Esters in Flowers and Damaged Leaves of Clarkia Breweri1

Characterization of an Acyltransferase Capable of Synthesizing Benzylbenzoate and Other Volatile Esters in Flowers and Damaged Leaves of Clarkia breweri1

John C. D’Auria, Feng Chen, and Eran Pichersky* Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048

A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile.

Floral scents are often rich in volatile esters. In agents to prevent further spread of disease (Hamil- Clarkia breweri ([Gray] Greene; Onagraceae), an an- tonkemp et al., 1992; Croft et al., 1993; Deng et al., nual plant native to California, for example, benzyl- 1993). acetate constitutes 20% to 40% (w/w) of the total We have previously reported the characterization scent output (depending on the particular C. breweri of the enzyme acetyl CoA:benzyl alcohol acetyl trans- line), whereas the two other esters present in its ferase (BEAT), which is responsible for the produc- aroma, benzylbenzoate and methylsalicylate, each tion of the floral volatile benzylacetate in C. breweri contribute about 5% to the total volatile output (Ra- flowers (Dudareva et al., 1998a). The concurrent guso and Pichersky, 1995). characterization of BEAT and several other structur- Many plants also emit volatile esters from leaves ally similar enzymes has led to the recognition of a damaged by herbivores. The most commonly re- novel class of evolutionarily related acyltransferases ported volatile esters in this class are those derived (EC 2.3.1.x) commonly referred to as the BAHD fam- from the octadecanoid fatty acids, such as cis-3- ily of acyltransferases (St-Pierre and De Luca, 2000). hexen-1-yl acetate (Ozawa et al., 2000; Arimura et al., There are approximately 60 BAHD gene family mem- 2001; Mattiacci et al., 2001), and methylsalicylate, bers in the model organism Arabidopsis, although mostly likely derived from the phenylpropanoid to date, the substrates and products of the enzymes pathway (Lee et al., 1995) or the shikimate pathway encoded by these genes have not been determined. (Wildermuth et al., 2001), is also common (Pare and Members of the BAHD family have been identified in Tumlinson, 1996; Van Poecke et al., 2001). Total vola- other plant species through expressed sequence tag tiles (including, but not limited to, esters) emitted (EST) database construction and analyses, and in a from injured leaves have been shown to function as few cases, the biochemical function has been deter- orientation cues for predatory wasps and mites that mined as well. For example, benzoyl-CoA:anthrani- feed upon herbivorous insects (Turlings et al., 1990; late N-benzo-yltransferase (HCBT) from carnation De Moraes et al., 1998). In addition, some of these (Dianthus caryophyllus) is expressed during infection volatiles may serve as antimicrobial or antifungal with Fusarium oxysporum or Phytophthora parasitica and produces several different benzoylated and cou- 1 This work was supported by the National Science Foundation maroylated anthranilide phytoalexin derivatives (grant no. MCB–9974463), by Novartis Agribusiness Biotechnology (Yang et al., 1997). Other BAHD proteins that are also Research, Inc., and by a National Institutes of Health training grant known to be involved in plant defense are TAT and fellowship in genetics to J.C.D. (grant no. 5 T32 GM07544). * Corresponding author; e-mail: [email protected]; fax 734–647– DBAT from Taxus cuspidata, which catalyze the acet- 0884. ylation and benzoylation, respectively, of taxol pre- Article, publication date, and citation information can be found cursors (Walker and Croteau, 2000a, 2000b; Walker et at www.plantphysiol.org/cgi/doi/10.1104/pp.006460. al., 2000), MAT and DAT from Catharanthus roseus,

466 Plant Physiology, September 2002, Vol. 130, pp. 466–476, www.plantphysiol.org © 2002 American Society of Plant Biologists Damage-Inducible Acyltransferases which catalyze the acetylation of precursors of the alkaloid vindoline (Power et al., 1990; St-Pierre et al., 1998; Laflamme et al., 2001), and SALAT, which is involved in morphine biosynthesis (Grothe et al., 2001). BAHD enzymes involved in caffeoylation and malonylation of anthocyanin pigments have also been reported (Fujiwara et al., 1997, 1998; Yonekura- Sakakibara et al., 2000; Suzuki et al., 2001) as well as those involved in volatile production in strawberry (Fragaria spp.) fruit (Aharoni et al., 2000). It should be noted that the enzymes responsible for the synthesis of methyl esters such as methylsalicylate and meth- yljasmonate belong to a different family of enzymes (Ross et al., 1999; Seo et al., 2001). Figure 2. BEBT enzymatic activity in different floral tissues during Here, we report the characterization of the gene the lifespan of the flower. Petal and stigma tissues were collected and enzyme for the biosynthesis of the floral volatile from C. breweri flowers daily starting 2 d before flower opening (d benzylbenzoate in C. breweri. The gene for benzoyl- Ϫ2) and ending on d 4 postanthesis. For each data point, tissues from CoA:benzyl alcohol benzoyl transferase (BEBT) is ex- three different plants were combined for each assay, at least three pressed in flowers, but it is also expressed in leaf independent assays were conducted, and the mean was obtained. F, tissue after damage. The structure of BEBT indicates Petals; f, stigma. pkat, Picomoles of product per second. that it belongs to the BAHD family of acyltransferases, and its wide substrate specificity may allow d 4, at which time BEBT levels were similar to those it to catalyze the formation of other esters as well. in the stigma (Fig. 2).

RESULTS Isolation of a cDNA Clone Encoding BEBT Benzylbenzoate-Forming Activity in Flowers We have recently undertaken the sequencing of We have previously shown that flowers of C. brew- approximately 750 EST cDNAs from a C. breweri eri emit benzylbenzoate (Raguso and Pichersky, flower tissue cDNA library. Because the acylation 1995). The emission of this volatile is highest from the reaction performed by BEBT is similar in principle to stigma, with petals contributing most of the rest the acylation reactions performed by BAHD-type en- (Dudareva et al., 1998b). Total floral emission peaks zymes, we searched the database with BAHD-type 36 h postanthesis, with a second minor peak 72 h protein sequences, including C. breweri BEAT and postanthesis. putative BAHD proteins from Arabidopsis, using the To look at the synthesis of benzylbenzoate, we BLAST2 program (Altschul et al., 1990). The search developed an assay (see “Materials and Methods”)to identified one cDNA with homology to BAHD acyl- detect the enzymatic activity of BEBT, the hypothet- transferase sequences. Because this cDNA was in- ical enzyme (Croteau, 1977; Dudareva et al., 1998b) complete on both the 5Ј and 3Ј ends, we performed that would catalyze the formation of benzylbenzoate 5Ј- and 3Ј-RACE experiments to obtain a full-length from benzyl alcohol and benzoyl CoA (Fig. 1). Crude clone. The complete cDNA, which we tentatively extracts of stigma and petal tissues from flowers of designated BEBT, has an open reading frame encod- different ages were assayed. BEBT activity was de- ing a protein with 456 amino acid residues and a tected in both petals and stigma. In the stigma, it was calculated molecular mass of 50.6 kD. Protein se- high already in unopened flowers, and it peaked on quence comparisons indicate that BEBT is most sim- d 1 of anthesis (Fig. 2). On d 2, when the stigma ilar (72% identity) to a protein encoded by the to- becomes receptive, there was a precipitous drop in bacco (Nicotiana tabacum) hypersensitive response BEBT activity of about 3-fold, and activity remained cDNA HSR201 (Czernic et al., 1996) and to two low on d 3 and 4. In petals, BEBT activity was much BAHD-like Arabidopsis proteins (protein ID nos. lower than in stigma but it gradually increased until CAC01898.1 and AAF01587.1) that are 57% and 54%

Figure 1. The reaction catalyzed by BEBT.

Plant Physiol. Vol. 130, 2002 467 D’Auria et al. identical, respectively (Fig. 3). In addition, proteins the sequencing of the rice genome are also very sim- encoded by a cDNA from cantaloupe (Cucumis melo) ilar to BEBT, being 53% and 63% identical, respec- and a gene from rice (Oryza sativa) identified during tively, to BEBT. The proteins encoded by these genes all share the salient features of the BAHD protein family, most notably the HXXXD motif in the center of the protein believed to be involved in catalysis, and the DFGWG motif of unknown function near the C terminus (St-Pierre and De Luca, 2000).

Enzymatic Characterization of C. breweri BEBT Expressed in Escherichia coli To determine whether the isolated C. breweri cDNA indeed encoded an enzyme with BEBT activity, we subcloned the complete open reading frame of BEBT into the expression vector pET11a, transformed E. coli cells with the recombinant vector, and induced the expression of BEBT with IPTG. Because E. coli strain BL21(DE3) pLysS, the standard strain for expressing heterologous genes from pET vectors, has a low level of acyltransferase activity derived from the chloram- phenicol O-acetyltransferase gene, we used E. coli strain B834 instead. Strain B834 cells have no activity with either acetyl CoA or benzoyl CoA regardless of whether they carry a pET expression vector and regardless of whether they have been treated with IPTG (data not shown). The spent media of BEBT-expressing E. coli cultures were extracted with pentane and analyzed by gas chromatography-mass spectroscopy (GC-MS), re- vealing a wide variety of acylated esters carrying acyl moieties ranging from C2-C6 (Table I). In contrast, spent media of induced cultures carrying a pET11a plasmid with no insert, used as a control, contained none of these esters. Because the ester composition in the spent media of BEAT-expressing E. coli cultures is Ͼ98% benzylacetate (Dudareva et al., 1998a), it indicates that BEBT, unlike BEAT, may accept several

Table I. Volatile esters extracted from the spent media of E. coli cells expressing BEBTa Esters Ester/Spent Medium ng mLϪ1 Acetyl n-Butyl acetate 5,576.8 Ϯ 947.8 n-Pentyl acetate 73.7 Ϯ 17.6 Benzyl acetate 568.8 Ϯ 139.9 Figure 3. Sequence comparisons of BEBT and BEAT from C. breweri, Phenethyl acetate 180.0 Ϯ 90.6 HSR201 from tobacco, and the two most similar proteins to BEBT Butyryl from Arabidopsis. The BEBT gene accession number is AF500200, Ethyl butyrate 680.6 Ϯ 285.5 and the gene accession number of HSR201 is AF500202. The two n-Butyl butyrate 1,658.3 Ϯ 326.4 Arabidopsis acyltransferase proteins in this figure are identified by Hexanoyl their protein accession numbers and correspond to genes from two Ethyl hexanoate 734.2 Ϯ 67.6 BAC clones (accession nos. AL391151 and AC009895, respectively). n-Butyl hexanoate 530.5 Ϯ 92.4 A, Protein sequence alignments of all five proteins using the ClustalX Others program. Amino acids shaded in black represent identical matches; n-Butyl propanoate 70.8 Ϯ 17.5 gray shaded boxes represent conservative changes. The HXXXD Ethyl octanoate 479.0 Ϯ 204.4 motif is indicated with arrowheads; the DFGWG motif is indicated a Ͼ with asterisks. B, Maximum parsimony tree based on protein se- Only the compounds that constituted 1% of the total volatiles present and that were not found in the controls are shown. quence alignments from A using the program PAUP*.

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Table II. Kinetic parameters of BEBT

Km Kcat Kcat/Km

Ϫ1 Ϫ1 Ϫ1 ␮M s nM s Benzyl alcohol 46.8 4 85.2 (with benzoyl CoA) Cinnamyl alcohol 97.8 4.6 47.2 (with benzoyl CoA) Benzoyl CoA 20.5 2.5 124 (with benzyl alcohol) Cinnamoyl CoA 464 0.4 0.9 (with benzyl alcohol) Acetyl CoA 818 17.5 21.5 (with benzyl alcohol)

Figure 4. Purification of C. breweri BEBT produced in E. coli. Lane 1, Mr markers. Lane 2, Q-Superose fraction with the highest levels of ␮m (Table II), strongly suggests that acetyl CoA is not BEBT-specific activity. commonly used by BEBT as the acyl donor in vivo. ␮ With benzoyl CoA, for which a Km value of 20.5 m acyl-CoA substrates in addition to acetyl-CoA. Al- was determined, BEBT could use benzyl alcohol though the products that formed in E. coli when (with a Km value of 46.8) and a variety of other BEBT was expressed clearly depended both on the alcohols, some of them with higher rates (at a sub- affinity of the BEBT enzyme to potential substrates strate concentration of 1 mm), particularly octanol and the availability of these substrates in E. coli and and cinnamyl alcohol (Table III). BEBT had a Km although the substrate availability and concentration value for cinnamyl alcohol of 97.8 ␮m (Table II). We in the plant cell are likely to be very different from also examined the activity of BEBT with other acyl the situation in E. coli, nonetheless these preliminary CoA substrates by performing competition experi- results demonstrated that BEBT possessed ester- ments (Beuerle and Pichersky, 2002a) of these nonra- forming activity. dioactive CoA esters with radioactive acetyl CoA. To characterize the biochemical properties of BEBT Benzoyl CoA and cinnamoyl CoA were better com- in vitro, we purified the complete, non-fusion BEBT petitors than either butanoyl CoA or hexanoyl CoA. enzyme from the crude E. coli extract by anion- The Km value for cinnamoyl CoA was determined to exchange chromatography on DEAE, followed by be 464 ␮m (Table II). Coumaroyl CoA, on the other another anion-exchange column, Mono Q, and finally hand, did not act as a competitor and could not serve by size-exclusion chromatography on a Q-Superose as a substrate. On the basis of the these results and column (Fig. 4), as described in “Materials and Meth- the measured Km values, we concluded that benzoyl ods” (we had originally tried to introduce a His tag at CoA and benzyl alcohol are likely to be in vivo either the N- or C-terminal ends of the protein, however both constructs yielded a protein with a dramatically lower activity). After the Q-Superose purifica- Table III. Relative activity of BEBT with a variety of substrates tion step, BEBT was essentially separated from all but Benzoyl CoA Acetyl CoA as one other protein with a molecular mass of 22.6 kD, Alcohol Substrate as the Acyl the Acyl which constituted about 40% of the total protein Substrate Substrate concentration. Although the latter protein was Benzyl alcohol 100a 1001 present in the Q-Superose fraction with the highest ␳-Hydroxy benzyl alcohol 0 0 BEBT activity, its concentration in this and adjacent m-Hydroxy benzyl alcohol 113 8.7 fractions did not correlate well with the levels of Cinnamyl alcohol 172.6 8.4 BEBT activities in these fractions, whereas BEBT pro- 2-Phenyl ethanol 56.9 12.1 tein concentrations and activity levels did (data not Anthranilate 0 0 cis-3-Hexen-1-ol 149.9 50.5 shown). It is noteworthy that BEBT migrated on SDS- Geraniol 190.8 45.4 PAGE as a protein with a molecular mass of 55 kD, Linalool 0 0.6 whereas its calculated molecular mass is 50.6 kD. Ethanol 0 0 BEAT protein similarly has a calculated molecular 1-Butanol 107.6 35.5 mass of 48.2 kD, but migrates on a gel as a protein Octanol 205.2 71.5 with a molecular mass of 58 kD (Dudareva et al., Hexanol 148.2 32.9 1998a). aActivity with benzylalcohol was set at 100%, and other activities In initial assays of BEBT activity, we used a variety are shown as a percentage of this activity. All alcohol substrates were of alcohols with benzoyl CoA and acetyl CoA. With tested at a concentration of 1 mM. The specific activity of BEBT with Ϫ acetyl CoA, benzyl alcohol was the preferred sub- benzoyl CoA and benzyl alcohol as substrates was 17.1 nkat mg 1 strate, but other alcohols could also be used (data not protein, and the specific activity of BEBT with acetyl CoA and benzyl alcohol as substrates was 1.4 nkat mgϪ1 protein. shown). However, the Km value for acetyl CoA, 818 Plant Physiol. Vol. 130, 2002 469 D’Auria et al.

petals had approximately the same amount of transcripts, although it was approximately 30% of that found in the stigma. Transcript levels in style tissue were 10% of those found in the stigma, whereas healthy leaves had little discernible expression of BEBT (Fig. 5). The levels of BEBT transcripts were examined over the life span of the flower by comparing the stigma, the organ with the highest levels of BEBT transcripts, to the petals, the largest organ (by mass) of the flower. The stigmata of flower buds 2 d before anthesis contained the highest levels of BEBT transcripts, and these levels subsequently dropped but

Figure 5. RNA gel-blot analysis of the relative abundance of BEBT mRNA transcripts in the stigma, stamen, style, sepals, petals, and leaves of C. breweri plants. Tissues were harvested from mature plants and floral tissue samples were taken from flowers ond1of anthesis. Lanes were loaded with 4 ␮g of total RNA. After probing with the BEBT probe and quantitation of the results, each blot was rehybridized with an 18S rDNA probe to normalize samples. A sample blot is shown above. The graphical representation below represents an average of three independent experiments. substrates of BEBT. It is possible that other alcohols similar to benzyl alcohol (such as octanol) and other acyl CoA esters similar to benzoyl CoA (such as cinnamoyl CoA) may also be acted upon by BEBT, but at present, we are not aware of other possible ester products in C. breweri besides benzylbenzoate that could result from catalysis by BEBT. The pH optima of BEBT was determined to be pH 7.7, whereas only 39% and 69% of maximal activity were observed at pH of 6.5 and of 9.0, respectively. The enzyme was 100% stable for 30 min at 30°C and 80% stable for 30 min at 37°C. After incubation at 50°C for 30 min, the enzyme was completely inacti- vated. The effects of monovalent and divalent cations were also tested. None were found to be stimulatory, ϩ ϩ ϩ ϩ ϩ however Mg2 ,Ca2 ,Co2 ,Zn2 , and Cu2 had a strong inhibitory effect (50%–100% inhibition at a final concentration of 5 mm). The apparent molecular mass of the protein as determined by size-exclusion chromatography was approximately 47 to 49.5 kD. Because the predicted molecular mass of the BEBT protein is 50.6 kD, we concluded that BEBT is a Figure 6. Expression of BEBT in petal and stigma tissues of C. breweri monomeric enzyme. flowers during floral development. A, RNA gel-blot analysis of the relative abundance of BEBT mRNA in stigma tissue. Stigma tissue was collected daily from flowers starting 2 d before flower opening and Organ Specificity and Developmental Changes of BEBT continuing until d 4 postanthesis. B, RNA gel-blot analysis of the relative Expression in C. breweri abundance of BEBT mRNA in petal tissue. Petal tissue was collected at the same times as the tissues in A. For all experiments, each lane was RNA gel-blot analysis with d 1 postanthesis flow- loaded with 4 ␮g of total RNA. After hybridization with the BEBT probe ers and leaf tissue was carried out to examine the and quantitation of the results, blots were stripped and reprobed with an tissue(s) in which the BEBT gene is expressed (Fig. 5). 18S rDNA probe to normalize samples. In each panel, a sample blot is The highest steady-state levels of BEBT transcripts shown above, and the graphical representation below represents an were observed in the stigma. Stamens, sepals, and average of two independent experiments.

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Characterizaton of the Activities of the Proteins Encoded by Tobacco HSR201 cDNA and by Another BEBT-Related cDNA from Arabidopsis The sequence of the protein encoded by the HSR201 cDNA from tobacco is the most similar to BEBT of the protein sequences currently found in the databases (Fig. 3). It has previously been shown that HSR201 is expressed in damaged leaves of tobacco after infection with the microorganism Pseudomonas solanacearum (Czernic et al., 1996). To analyze the function of the protein encoded by HSR201, we isolated total RNA from tobacco leaves 6 h after dam- aging them with a razor blade, and cloned the open reading frame of HSR201 into the pCR T7 TOPO-CT Figure 7. Variation of the levels of BEBT protein over the lifespan of expression vector, using oligonucleotides based on the stigma. Stigma tissue was collected daily from flowers starting 2 d the published sequence. The HSR201 protein ob- before flower opening, and continuing until d 4 postanthesis. Sam- tained in the E. coli expression system had BEBT ples were run on SDS-PAGE, and the gels were blotted onto filters. activity with Km values for benzoyl CoA and benzyl The filters were first probed with anti-BEBT antibodies, followed by alcohol of 35 and 19 ␮m, respectively, similar to the incubation with secondary antibodies conjugated to alkaline phos- corresponding Km values of BEBT. The HSR201 pro- phatase. Bands were visualized by chemiluminescence. A sample tein showed no activity with acetyl CoA at concen- blot is shown above, and the graphical representation below repre- trations Ͻ1mm. sents an average of two independent experiments. We also examined the activities of the proteins encoded by the two Arabidopsis genes whose proteins show the highest similarity to BEBT. cDNAs remained within 40% to 60% of their initial value by encoding CAC01898.1 and AAF01587.1 were ex- d 3 to 4 postanthesis (Fig. 6A). In contrast, BEBT pressed in E. coli. No activity could be detected mRNA transcript levels from petal tissue steadily for protein CAC01898.1 with a variety of alcohols increased throughout the lifespan of the flower, reaching a 5-fold increase on d 4 postanthesis as compared with d Ϫ2 (Fig. 6B). However, BEBT mRNA levels in petals at any given day were always severalfold lower than those in the stigma.

Quantitation of BEBT Protein Levels in the Stigma by Immunoblotting BEBT protein levels were quantified in the stigma by SDS-PAGE followed by immunoblots of stigma samples together with samples of known amounts of BEBT purified from E. coli (Fig. 7, top). In stigma extracts from all stages of development, with the exception of d Ϫ2 stigma, the antibodies made against purified C. breweri BEBT produced in E. coli (see “Materials and Methods”) recognized a single protein that migrated on the gel to the same position Ϫ as did BEBT produced in E. coli.Ind 2 stigma, the Figure 8. RNA gel-blot analysis of the relative expression of BEBT in anti-BEBT antibodies recognized an unfocused pro- wounded and non-wounded leaf tissues of C. breweri. Mature leaves tein band migrating slightly more slowly than E. were harvested either before (untreated) or after mechanical wound- coli-produced BEBT (Fig. 7, top). The reasons for the ing. Leaves were wounded by making two parallel incisions approx- aberrant behavior of BEBT in d Ϫ2 stigma in SDS- imately 8 mm long to each side of the midvein with a sterile razor PAGE could not be determined. The quantitative blade, and tissue was collected at 0, 1, 2, 4, 6, 8, 12, and 24 h after results of the immunoblots (Fig. 7, bottom) indicated wounding. RNA from stigma tissue (included for comparison) was collected from d 1 flowers. Total RNA (4 ␮g) was loaded onto each that BEBT protein levels followed the measured lev- lane of the gel. After probing with the BEBT probe and quantitation els of BEBT enzymatic activity (Fig. 2), dropping of the results, the blots were then stripped and reprobed with an 18S equally rapidly on d 2 postanthesis, the day when the rDNA probe to normalize samples. A sample blot is shown above, stigma lobes open up and the stigma becomes recep- and the graphical representation below represents an average of two tive, compared with d 1 postanthesis. independent experiments.

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and either acetyl CoA or benzoyl CoA. Protein substrates, and they strongly argue that BEBT is re- AAF01587.1 had no detectable activity with benzoyl sponsible for the synthesis of benzylbenzoate, at least CoA at concentrations Ͻ1mm but exhibited strong in flowers. For comparison, the enzyme benzoyl- activity with acetyl CoA and cis-3-hexen-1-ol and re- CoA:taxane 2␣-O-benzoyltransferase from Taxus spp. ␮ lated alcohols (e.g. hexanol and octanol). AAF01587.1, was reported to have a Km value of 300 m for which we have designated CHAT (for acetyl CoA:cis- benzoyl CoA (Walker and Croteau, 2000b). The en- 3-hexen-1-ol acetyl transferase) had a Km value of 10.5 zyme HCBT, another BAHD benzoyltransferase, has ␮ ␮ ␮ m for acetyl CoA and 165 m for cis-3-hexen-1-ol. a Km value of 50 m for benzoyl CoA, similar to that of BEBT (Yang et al., 1997). There is currently no data BEBT Expression in Wounded Leaves on the concentrations of benzoyl CoA in C. breweri or any other species known to make benzoate esters, Because HSR201 proved to encode BEBT and its although benzoic acid:CoA ligase has recently been expression was known to be induced in leaves upon identified and partially purified from C. breweri wounding, we examined the expression of C. breweri (Beuerle and Pichersky, 2002a). On the other hand, BEBT in wounded leaves. Total RNA was extracted the Km values of BEBT for acetyl-CoA and cinnamoyl from damaged leaves at intervals of 0, 1, 2, 4, 6, 8, 12, CoA—818 ␮m and 464 ␮m, respectively—suggest and 24 h after wounding, and the RNA samples were that these acyl CoAs are not commonly used by analyzed by gel blotting and probing with a BEBT BEBT. In addition, the ratio of Kcat/Km (benzoyl CoA) probe (Fig. 8). BEBT mRNA levels rose dramatically to Kcat/Km (acetyl CoA) shows that BEBT preferentially from 2 to 6 h after wounding, with peak expression uses benzoyl CoA to acetyl CoA by a factor of 5.8. evident after 6 h, decreasing afterward. It should be Additional support for the role of the protein en- noted that BEBT transcript levels in wounded leaves coded by the BEBT gene in benzylbenzoate produc- were still roughly one-half of that found in the stigma tion in flowers comes from the general correlation of d 1 flowers. between BEBT mRNA levels and BEBT enzyme activity in the different floral parts (Figs. 2 and 4) and DISCUSSION from the correlation between BEBT enzymatic activity in the stigma and the amount of BEBT protein as BEBT, a BAHD-Type Acyltransferase, Is Responsible for determined by immunoblotting with antibodies Benzylbenzoate Synthesis in C. breweri Flowers made against the protein encoded by the BEBT gene The BAHD family of acyl transferases is a recently (Figs. 2 and 6). discovered group of enzymes (St-Pierre and De Luca, Although BEBT transcript levels and BEBT enzy- 2000). The function and substrate specificity of only a matic activity levels in the stigma are always higher few representatives, which include enzymes that cat- than those in the petals, nonetheless the levels of alyze the formation of both volatile and nonvolatile BEBT enzymatic activity in the stigma show an inter- esters, have so far been determined. Even so, it is esting pattern of change relative to the BEBT mRNA clear that a hallmark of these enzymes is the rela- levels. BEBT activity levels show a moderate buildup tively wide range of alcohols that they can react with. untild1ofanthesis followed by quick decline, even On the other hand, the range of acyl-CoA substrates though BEBT mRNA levels do not show such a cor- has not yet been extensively tested, due mostly to the responding decline. The buildup in BEBT activity lack of availability of these compounds. However, at levels untild1ofanthesis suggests that the turn-over least in one case, that of HCBT from carnation, it was of the BEBT protein is slow (as is the case for other scent enzymes (Dudareva and Pichersky, 2000), and shown that the enzyme has a somewhat lower Km value for cinammoyl CoA than for benzoyl CoA, therefore BEBT protein continues to accumulate even even though benzoyl CoA is the substrate used in though BEBT steady-state transcript levels are rela- vivo (Yang et al., 1997). tively stable. The precipitous decline in BEBT enzy- The products detected in the spent media of E. coli matic levels (per gram fresh weight) ond2ofanthe- cells expressing BEBT (Table I) show that BEBT can sis is clearly attributable to a decrease in total BEBT use several medium-chain acyl CoA substrates and a protein (Fig. 7), and this decline is not caused by a variety of mostly primary alcohols in addition to similar decline in the steady-state levels of BEBT benzoyl CoA and benzyl alcohol. However, what mRNA (Fig. 6A). Day 2 of anthesis is the day in products are formed in E. coli depend on the presence which the stigma opens up and becomes receptive, a and concentrations of the respective substrates vis- process that involves rapid cell expansion and con- a`-vis the affinity of BEBT for those substrates. The sequently a reduction in protein content per cell possibility that the in planta substrates of BEBT are (Pichersky et al., 1994). However, even when BEBT not found in E. coli is highly likely. activity levels and BEBT protein levels are plotted The Km values of BEBT for the substrates benzyl after normalization to protein content (data not alcohol and benzoyl CoA—46.8 and 20.5 ␮m, respec- shown), a substantial drop of 50% in both is still tively—are in a range that is similar to those of BEAT observed fromd1to2.This reduction in BEBT and several other BAHD acyltransferases for their activity, therefore, must involve a mechanism of spe-

472 Plant Physiol. Vol. 130, 2002 Damage-Inducible Acyltransferases

cific protein degradation and may be related to the (Sunshine mix no. 2, Sun Gro Horticulture, Bellevue, WA) and grown under yet-undetermined function of benzylbenzoate in the 12-h light/12-h dark light conditions in a growth chamber. stigma. Reagents

Acyltransferase Activities in Wounded Leaves All solvents and reagents were molecular biology grade or reagent grade and were obtained from Sigma (St. Louis), Aldrich (Milwaukee), or Fisher It is well established that fatty acid-derived alde- Scientific (Loughborough, Leicestershire, UK). Radiolabeled [acety-1-14C]CoA was purchased from ICN (Irvine, CA). [7-14C]Benzoyl-CoA was enzymati- hyde, alcohol, and ester “green-leaf” volatiles are Ϫ emitted upon injury (Walling, 2000). Our results cally synthesized from [7-14C]benzoic acid (16.6 mCi mmol 1; Sigma) as showing the induction of BEBT in wounded leaves previously described (Beuerle and Pichersky, 2002b). and the induction of Arabidopsis CHAT by wounding (J.C. D’Auria, unpublished data) point to the Enzyme Extraction and Assay possible role of such acetyltransferases in the defense Crude protein extracts were prepared by macerating tissue parts in a response of leaves to injury. Although CHAT may be microcentrifuge tube with a Teflon-coated homogenizer in the presence of involved in the biosynthesis of cis-3-hexenyl acetate, ice-cold buffer containing 50 mm Bis-Tris-HCl, pH 7.0, 5 mm dithioerythritol it is likely that BEBT activity in the leaf is responsible (DTE), 1% (w/v) polyvinylpyrrolidone-40, and 10% (v/v) glycerol. A ratio mostly for the production of benzylbenzoate. Other of extraction buffer:tissue fresh weight of 10:1 (v/w) was used. The slurry was then centrifuged for 10 min at 4°C, and the supernatant was transferred species are also known to produce this compound in to a new tube. For each time point, flower tissue and leaves from three leaves. For example, leaves of rice produce benzyl- different plants were combined, and assays were repeated at least twice. benzoate in response to oviposition by the insect Assay samples were prepared by adding the following to a 0.6-mL whitebacked planthopper (Sogatella furcifera), and microcentrifuge tube: 5.0 ␮L of crude extract, 10.0 ␮Lof5ϫ assay buffer (250 ␮ it was shown that benzylbenzoate has ovicidal prop- mm Bis-Tris Propane, pH 7.7), 1.0 Lof50mm alcohol substrate dissolved in dimethyl sulfoxide, 1.0 ␮Lof[7-14C]benzoyl-CoA, and 33 ␮L of water to erties (Seino et al., 1996). Our results showing that bring the assay volume to 50 ␮L. Acetyltransferase assays for both BEBT and the tobacco hypersensitive-response cDNA HSR201 CHAT were performed as above with the substitution of 0.25 ␮L of [acetyl- encodes BEBT suggest that tobacco leaves may also 1-14C]CoA (50 mCi mmolϪ1) for the radiolabeled benzoyl-CoA. The assays synthesize benzylbenzoate or a related compound were carried out at room temperature for 30 min. The radioactively labeled acylated product was extracted twice with 180 ␮L of hexane, and the total under stress. Other esters that are known to be organic phase was transferred to a scintillation vial with 2 mL of nonaque- synthesized in damaged leaves include ␳-coumaroyl- ous scintillation fluid (Econo-Safe, Research Products International, Mount and feruloyltyramine in tomato (Lycopersicon esculen- Prospect, IL) and counted in a liquid-scintillation counter (model LS6500, tum; Pearce et al., 1998) and 5-caffeoylquinic (chloro- Beckman Coulter, Fullerton, CA). The raw data (counts per minute) were genic) acid and its derivatives in lettuce (Lactuca converted to picokatals based on the specific activity of the radiolabeled substrate and the known efficiency of the scintillation counter used. Assays sativa; TomasBarberan et al., 1997). It is worth noting in which no alcohol substrate was added were performed to test back- that RNA-blot analysis indicated that the BEBT- ground thioesterase activity. In addition, boiled enzyme extracts were sub- related gene BEAT, which encodes an enzyme that stituted for intact enzyme to test nonenzymatic breakdown of the CoA catalyzes the formation of benzyl acetate, is not gen- thioester via reaction with reducing agents in the enzyme extract. The identity of the products were verified by GC-MS as described previously erally expressed in C. breweri leaves (Dudareva et al., using authentic standards (Dudareva et al., 1998a). 1998a) nor is it induced upon damage (N. Dudareva and E. Pichersky, unpublished data). BEBT cDNA Identification

Evolution of BEBT and CHAT A total of 735 cDNAs were randomly and automatically isolated and sequenced similar to those previously reported for a basil (Ocimum basili- Although BEBT and HSR201 have strong affinity to cum) EST database (Gang et al., 2001). The C. breweri flower EST database can be found at: https://sativa.biology.lsa.umich.edu/blast/blast.html benzoyl CoA and low affinity to acetyl CoA, they are (login, clarkia; password, breweri). The BEBT cDNA was identified by closely related to CHAT, which has a much stronger BLAST searches with other BAHD sequences. affinity to acetyl CoA than to benzoyl CoA. A similar situation was reported for two closely related acyltransferases from Taxus spp., one that uses acetyl Cloning of BEBT and Its Homologs from CoA and the other benzoyl CoA (Walker and Cro- Arabidopsis and Tobacco teau, 2001). This suggests that the specificity for the The BEBT cDNA clone identified in the C. breweri EST database was acyl CoA moiety can evolve relatively easily in the incomplete. RACE was used to obtain the missing sequence from either end BAHD family of enzymes. (Czernic et al., 1996; Martz et al., 1998). Two specific primers were designed (forward primer, 5Ј-CCCATTCGACGACCTGGCTCACCGG-3Ј; backward primer, 5Ј-CCCAGCTCGTGTCACGTCTGAAACCAAG-3Ј) and used in 5Ј- MATERIALS AND METHODS and 3Ј-RACE, respectively. First-strand cDNAs for the RACE experiments were made from total RNA of 1-d-old C. breweri flowers. On the basis of the Plant Material and Growth Conditions sequences obtained, the full-length cDNA was amplified from first-strand cDNA using the forward primer 5Ј-AATGGCGCATGATCAATCTCTCT-3Ј Details of the construction of true-breeding Clarkia breweri stocks and and the backward primer 5Ј-TCTCATCAAAGGGAAGACTGTGT-3Ј.The growth conditions are as described (Raguso and Pichersky, 1995). Arabi- resulting 1,375-nucleotide (nt) DNA fragment was subcloned into a TA dopsis ecotype Columbia plants were used of for cloning CHAT. Tobacco cloning vector (pCRT7/CT TOPO-TA, Invitrogen, Carlsbad, CA). The se- (Nicotiana tabacum) seeds were germinated on Murashige and Skoog me- quences of independent clones were determined in their entirety on both dium plates and grown for 2 weeks. Seedlings were then transplanted to soil strands. For further expression analysis, the open reading frame of

Plant Physiol. Vol. 130, 2002 473 D’Auria et al.

BEBT was subcloned into the pET-T7(11a) expression vector (Novagen, was located. Antibodies were prepared by Cocalico Biologicals (Reamstown, Madison, WI). PA) by injecting macerated gel fragments containing the purified BEBT and For the gene residing on BAC clone AC009895 (CHAT), a full-length following the company’s protocol. Protein gel blots and quantitation of cDNA was obtained by RT-PCR using total RNA extracted from Arabidop- results were performed as described in Dudareva et al. (1996). sis flower tissue using the forward primer 5Ј-AATGGACCATCAAGTGTC- TCTGC-3Ј and the backward primer 5Ј-TCATCCTTTAGACACATT- TAGCACTCC-3Ј. The resulting 1,366-nt DNA fragment was cloned as Extraction of Volatile Esters from the Spent Media of described above for BEBT. All attempts to clone the gene residing on the E. coli and GC-MS Analysis BAC clone AL391151 via RT-PCR using total RNA from various plant tissues were unsuccessful. Because this gene has only one intron, the puta- Extraction of volatile esters from the spent media of E. coli expressing tive open reading frame was amplified from genomic DNA, and the introns BEBT or containing pET11a vector without any insert were performed and were spliced out as described by Nam et al. (1999) by using the following analyzed by GC-MS as reported by Dudareva et al. (1998a) with the substi- two pairs of primers (front pair, forward primer 5Ј-AATGTCCGGGT- tution of pentane for hexane as the solvent. CACTCACGTT and backward primer 5Ј-GCATTTCAAGCGCGTGA- CCTGCATGAGCAT-3Ј; back pair, forward primer 5Ј-CCTTTGATGC- TCATGCAGGTCACGCGCTTGAAATGC-3Ј and backward primer Molecular Mass Estimation 5Ј-TTACAGAGAAGACATGATCAA-3Ј). The resulting 1,387-nt DNA frag- Peak BEBT activity from the Mono-Q fractions was pooled, concentrated ment was again cloned as described above. down to 200 ␮L, and run on a gel filtration column (Q-Superose, Amersham The full-length cDNA clone HSR201 from tobacco was cloned by RT-PCR Pharmacia Biotech AB) precalibrated with molecular mass markers as pre- using first-strand cDNA obtained from total RNA extracted from mechan- viously described (Ross et al., 1999). ically wounded tobacco leaves 6 h after wounding. Wounds were made by making two lateral incisions to each side of the midvein with a sterile razor blade. The primers used in the RT-PCR were the forward primer Determination of Kinetic Parameters for 5Ј-AATGGATTCAAAGCAATCATCAGA-3Ј and the backward primer 5Ј- TCAAAGGGCAGGTCTGATAATG-3Ј and resulted in a DNA fragment that BEBT and CHAT was 1,384 nt in length. This fragment was also cloned into a TA cloning In all kinetics studies, appropriate enzyme concentration and incubation vector (pCRT7/CT TOPO-TA, Invitrogen) for expression analysis. times were chosen so that the reaction velocity was linear during the

incubation time period. To determine the Km value for each substrate, one Sequence Alignment and Phylogenetic Analysis substrate concentration was fixed at a saturated level and the concentration of the other substrate to be measured was varied. Lineweaver-Burk, Eadie-

Amino acid sequence alignments were generated using the ClustalX Hofstee, and Hanes plots were constructed and an average of their Km computer program (Thompson et al., 1997). Aligned sequences were ana- values were used to report the apparent Km value. lyzed using the PAUP* program (Phylogenetic Analysis Using Parsimony, Sinaur Associates, Sunderland, MA), with amino acids treated as unordered characters, and resulted in a single shortest tree shown in a PAUP* phylo- Temperature Effect on BEBT Activity gram format. A neighbor-joining tree was also generated. It showed the BEBT protein from the Mono-Q fraction was incubated at temperatures same branches as that in the maximum-parsimony tree. ranging from 4°Cto65°C for 30 min and then chilled on ice. Samples incubated at each temperature were then used for the enzyme assays. At Protein Production in Escherichia coli and least four independent assays were performed for each point, and an average was taken. BEBT Purification

BEBT expression in E. coli was induced as previously described (Nam et pH Optimum of BEBT Stability al., 1999) with the following minor modifications: All expression constructs in this study were transformed into the E. coli cell line B834. E. coli cells were The optimum pH for BEBT activity was determined using Bis-Tris pro- grown to an OD600 of 0.4 and then induced with 1 mm IPTG. After harvest- pane as the buffering system ranging from 6.5 to 9.0. Results presented are ing, the cells were resuspended in lysis buffer containing 50 mm Tris-HCl an average of at least four independent assays. (pH 8.0), 10 mm NaCl, 1 mm EDTA, 5 mm DTE, 1 mm phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol, lysed by sonification (3ϫ for 30 s), and centrifuged at 10,000g for 15 min. Effectors Soluble BEBT protein was first loaded onto DEAE anion-exchange column (10 mL of DE53, Whatman, Clifton, NJ) pre-equilibrated with buffer A Enzyme assays were performed with one of the following cations present ϩ ϩ ϩ ϩ (50 mm Bis-Tris, pH 7.5, 10% [v/v] glycerol, and 5 mm DTE) at a flow rate in the assay buffer at a final concentration of 5 mm:K ,Na ,Ca2 ,Cu2 , ϩ ϩ ϩ ϩ ϩ of1mLminϪ1 on an FPLC apparatus (Amersham Pharmacia Biotech AB, Fe2 ,Mg2 ,Mn2 ,Zn2 , and Co2 . Results presented are an average of at Uppsala). After washing off unbound materials from the column with buffer least four independent experiments. A, BEBT was eluted with a linear gradient of 0 to 0.5 mm KCl in buffer A. BEBT activity eluted in the 0.25 to 0.3 mm range. Fractions with the highest BEBT activity (10 mL) were pooled and diluted with buffer A to a final RNA Extraction and Northern-Blot Analysis volume of 40 mL and loaded onto a Mono-Q FPLC column (0.5-cm diam- eter ϫ 6.0 cm; Amersham Pharmacia Biotech AB). BEBT was eluted with a Total RNA was isolated from both C. breweri and Arabidopsis tissues 100 to 500 mm KCl linear gradient with 3-mL fractions being collected. BEBT using the RNeasy plant mini kit (Qiagen), using buffer RLC for C. breweri activity eluted with 130 to 140 mm KCl. Peak fractions from Mono-Q were and RLT for Arabidopsis. For the leaf-wounding treatments of C. breweri examined by SDS-PAGE gel electrophoresis followed by Coomassie Brilliant leaves, two parallel incisions of 8 to 10 mm were made on either side of the Blue or silver staining of the gel. BEBT protein concentration was deter- midvein using a sterile razor blade. Leaf tissue was collected at 0, 1, 2, 4, 6, mined as previously described (Bradford, 1976). 8, 12, and 24 h after the initial incision. In all cases, 100 mg of tissue was used, and the protocol described in the kit was followed. To reduce the amounts of polysaccharides extracted from C. breweri leaves using the RNeasy ␮ Preparation of BEBT Antibodies and Protein Gel Blots kit, 20 L of 50% high Mr PEG (15,000–20,000) was added to the microcentrifuge tube after the RLC buffer was added. This mixture of tissue, buffer, C. breweri BEBT protein produced in E. coli was purified from inclusion and PEG was incubated for 10 min at room temperature. After this, all steps bodies as previously described (Dudareva et al., 1996). The final step in the were performed as directed in the kit manual. purification consisted of electrophoresis on SDS-PAGE gel, staining with Total RNA (4 ␮g) from C. breweri tissues was resolved on 1% (w/v) Coomassie Blue, and excision of the gel segment where the separated BEBT agarose-formaldehyde gels, blotted to Hybond-Nϩ nylon membranes, hy-

474 Plant Physiol. Vol. 130, 2002 Damage-Inducible Acyltransferases

bridized, and stripped as described by Sambrook et al. (1989) and as per the Gang DR, Wang JH, Dudareva N, Nam KH, Simon JE, Lewinsohn E, ϩ instructions in the Hybond-N manual (Amersham Pharmacia Biotech AB). Pichersky E (2001) An investigation of the storage and biosynthesis of Probes were synthesized using the rediprime II kit (Amersham Pharmacia phenylpropenes in sweet basil. Plant Physiol 125: 539–555 Biotech AB) from a PCR-amplified fragment of BEBT using the full-length Grothe T, Lenz R, Kutchan TM (2001) Molecular characterization of the primers used previously for cloning. Hybridization signals were counted in salutardinol 7-O-acetyltransferase involved in morphine biosynthesis in a phosphor imager (Bio-Rad), stripped, and reprobed with a 18S rRNA opium poppy Papaver somniferum. J Biol Chem 276: 30717–30723 probe to normalize mRNA levels and control for discrepancies in concen- Hamiltonkemp TR, McCracken CT, Loughrin JH, Andersen RA, Hilde- tration readings given by spectrophotometer. brand DF (1992) Effects of some natural volatile compounds on the pathogenic fungi Alternaria alternata and Botrytis cinerea. J Chem Ecol 18: 1083–1091 ACKNOWLEDGMENTS Laflamme P, St-Pierre B, De Luca V (2001) Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19- We thank Dr. Ulrich Matern for providing us with a small amount of hydroxy-O-acetyltransferase. Plant Physiol 125: 189–198 [14C]benzoyl CoA used in the initial characterization of BEBT and Drs. Till Lee HI, Leon J, Raskin I (1995) Biosynthesis and metabolism of salicylic Beuerle and Yoko Sekiwa for the synthesis of [14C]benzoyl CoA and several acid. Proc Natl Acad Sci USA 92: 4076–4079 other substrates used in this study. 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