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First published online July 30, 2004; 10.1104/pp.104.045468 Plant Physiology 135:1993-2011 (2004) © 2004 American Society of Plant Biologists Understanding in Vivo Benzenoid Metabolism in Petunia Petal Tissue1Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 (J.B., F.N., X.C., C.M.K., B.W., G.P., I.O., D.R., N.D.); and Department of Plant Sciences and Institute for Biomedical Science and Biotechnology, University of Arizona, Tucson, Arizona 85721 (D.G.)
In vivo stable isotope labeling and computer-assisted metabolic flux analysis were used to investigate the metabolic pathways in petunia (Petunia hybrida) cv Mitchell leading from Phe to benzenoid compounds, a process that requires the shortening of the side chain by a C2 unit. Deuterium-labeled Phe (2H5-Phe) was supplied to excised petunia petals. The intracellular pools of benzenoid/phenylpropanoid-related compounds (intermediates and end products) as well as volatile end products within the floral bouquet were analyzed for pool sizes and labeling kinetics by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Modeling of the benzenoid network revealed that both the CoA-dependent,  -oxidative and CoA-independent, non--oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non--oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, -oxidative pathway. Modeling of 2H5-Phe labeling data predicted that in addition to benzaldehyde, benzylbenzoate is an intermediate between L-Phe and benzoic acid. Benzylbenzoate is the result of benzoylation of benzyl alcohol, for which activity was detected in petunia petals. A cDNA encoding a benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase was isolated from petunia cv Mitchell using a functional genomic approach. Biochemical characterization of a purified recombinant benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase protein showed that it can produce benzylbenzoate and phenylethyl benzoate, both present in petunia corollas, with similar catalytic efficiencies.
Phenylpropanoid metabolism comprises a complex series of branching biochemical pathways that provide plants with thousands of compounds, which are often specific to particular plant species. Many are intermediates in the synthesis of structural cell components (e.g. lignin, suberin, and other cell wall-associated phenolics), while others comprise a diverse array of pigments (e.g. flavonoids and anthocyanins), both of which are usually nonvolatile. However, phenylpropanoids that are reduced at the C9 position (to aldehyde, alcohol, or alkane/alkene) and/or that contain alkyl additions to the hydroxyl groups of the benzyl ring or to the carboxyl group (i.e. ethers and esters) are volatile. In addition, many benzenoid compounds, which lack the three-carbon chain and originate from trans-cinnamic acid (CA) as a side branch of the general phenylpropanoid pathway, are also volatile. These volatile phenylpropanoids/benzenoids are common constituents of floral scent (Knudsen et al., 1993  ). Recently, several enzymes that catalyze the formation of floral benzenoid volatiles have been identified and characterized. They include acetyl-CoA:benzyl alcohol acetyltransferase (BEAT; Dudareva et al., 1998a, 1998b), S-adenosyl-L-Met:salicylic acid carboxyl methyltransferase (Dudareva et al., 1998b; Ross et al., 1999; Negre et al., 2002), S-adenosyl-L-Met:benzoic acid carboxyl methyltransferase (Dudareva et al., 2000; Murfitt et al., 2000), benzoyl-CoA:benzyl alcohol benzoyl transferase (BEBT; D'Auria et al., 2002), and orcinol O-methyl transferase (Lavid et al., 2002), which are responsible for the formation of benzylacetate, methylsalicylate, methylbenzoate, benzylbenzoate, and dimethoxytoluene, respectively. In addition, S-adenosyl-L-Met:(iso)eugenol O-methyltransferase, which catalyzes the final step in the formation of the volatile phenylpropene, methyleugenol, was also isolated and characterized (Wang et al., 1997; Wang and Pichersky, 1998).
The biochemistry and enzymology of floral scent have mainly concentrated on the isolation and characterization of enzymes and genes involved in the final steps of the biosynthesis of scent volatile compounds. Surprisingly, little is known about the biochemical pathways leading to simple benzenoid compounds. The first committed step in the biosynthesis of benzenoid compounds is catalyzed by the well-known and widely distributed enzyme L-Phe ammonia-lyase (PAL; EC 4.3.1.5), just as in the biosynthesis of other phenylpropanoids. PAL catalyzes the deamination of L-Phe to produce CA. Formation of benzenoid compounds from CA requires the shortening of the side chain by a C2 unit, for which several routes have been proposed. The CoA-dependent,
The alternative CoA-independent, non- -oxidative pathway involves hydration of the free CA to 3-hydroxy-3-phenylpropionic acid (3-hydroxy-3-phenylpropanoic), side chain degradation via a reverse aldol reaction with formation of benzaldehyde, and its oxidation to benzoic acid (Fig. 1). A non--oxidative mechanism was demonstrated in three independent in vitro studies carried out with cell-suspension cultures of carrot (Schnitzler et al., 1992) and cell-free extracts of Lithospermum erythrorizum (Yazaki et al., 1991) and potato (French et al., 1976) for the formation of p-hydroxybenzoic acid, a key intermediate in several biosynthetic pathways, including biosyntheses of ubiquinones and the red naphtoquinone pigment shikonin. This non--oxidative pathway is characterized by the presence of p-hydroxybenzaldehyde as an important metabolic intermediate before oxidation to p-hydroxybenzoic acid and requires an aldehyde dehydrogenase for the conversion of an aldehyde to its corresponding carboxylic acid. A NADP+-dependent aldehyde dehydrogenase, which catalyzes the oxidation of coniferaldehyde and sinapaldehyde to ferulic and sinapic acids, respectively, was recently isolated and characterized from Arabidopsis (Nair et al., 2004).
The side chain shortening could also occur via a combination of these two pathways and could be CoA-dependent and non-
In this study, we use in vivo stable isotope labeling and metabolic flux analysis in combination with the power of computer-assisted metabolic modeling to investigate the metabolic pathways leading to benzenoid compounds in petunia (Petunia hybrida) cv Mitchell, a member of the Solanaceae family. Petunia cv Mitchell provides an ideal experimental system to investigate the benzenoid network because its floral scent consists almost exclusively of benzenoid/phenylpropanoid-related compounds (dominated by methylbenzoate, benzaldehyde, and phenylacetaldehyde), the levels of which change rhythmically through a daily light/dark cycle with a maximum at midnight (Kolosova et al., 2001
Experimental Design
Our previous results show that emission of methylbenzoate, one of the major scent compounds in petunia cv Mitchell flowers, changes rhythmically through a daily light/dark cycle with a maximum level at night (Kolosova et al., 2001
Although multiple mechanisms have been proposed for benzenoid biosynthesis from CA in plants, the first committed step in each of these pathways is the conversion of L-Phe to CA by PAL. Thus, in our labeling experiments we used deuterium ring-labeled Phe (2H5-Phe) supplied to excised petunia corolla limbs, a tissue highly specialized for floral scent biosynthesis that is able to continue producing and emitting volatile compounds after excision. Since petunia cv Mitchell is a nocturnally emitting plant (Kolosova et al., 2001 ; Verdonk et al., 2003), all feeding experiments were performed under dark conditions. To minimize the contribution of volatile and nonvolatile compounds from unlabeled areas of petal tissue and to limit the effect of natural rhythmic emission fluctuations during the daily light/dark cycle (Kolosova et al., 2001), quick uniform labeling of tissue was essential in these experiments. To find the optimal concentration of Phe for rapid uniform labeling, excised limbs of corolla were fed for 1 h through the cut edge with one of three different amounts of unlabeled Phe (0 µmol as a control, 37.5 µmol, and 375 µmol) combined with a small amount of radiolabeled Phe (2 µL of U-[14C]Phe). Exposure of petal tissues to x-ray film showed that feeding of 375 µmol Phe gave a more uniform distribution of labeled precursor resulting in 70% of the tissue labeled within an hour (Fig. 3A). Also, the amounts of emitted volatiles were slightly lower but comparable to the amounts emitted from flowers attached to the plant. Thus, this amount of Phe was used for further detailed time-course studies, including 30-, 60-, 120-, and 240-min labeling periods to ensure rapid label incorporation into metabolites. This level of exogenous Phe caused a large expansion of the endogenous Phe pool (Fig. 3B), and so it is likely that the flux measurements represent the maximal velocities of the metabolic system in vivo. Interestingly, when excised petunia limbs were not supplied with exogenous Phe, the emission levels of volatile benzenoid compounds were drastically decreased, indicating that petal tissue was not able to emit volatile compounds due to a limiting pool of endogenous Phe. Indeed, when the endogenous pool of Phe was measured in petal tissue at excision and 30 min later, we found that the endogenous pool of Phe rapidly declined from 445 ± 1.47 nmol g fresh weight (FW)–1 to 257 ± 79 nmol g FW–1 (Fig. 3B).
Since exogenously supplied precursors can affect the natural balance of cellular intermediates and increase the flux through the pathways, we investigated the changes in Phe pool levels in petal tissue after 30, 60, 120, and 240 min of feeding and the effect of the resulting endogenous Phe pool on expression and activity of PAL, the first enzyme in the network, and S-adenosyl-L-Met:benzoic acid/salicylic acid carboxyl methyltransferase (BSMT), the enzyme in petunia responsible for the biosynthesis of methylbenzoate (Negre et al., 2003 ), one of the end products in the network (Fig. 1). Previously, it has been shown that exogenously supplied Phe up-regulates the expression of some genes of the phenylpropanoid pathway, including PAL, in cell suspension culture (Anterola et al., 2002). Analysis of Phe levels in petal tissue after feeding revealed that the endogenous pool of Phe increased linearly almost 30 times by the end of experiment, from about 445 ± 1.47 nmol g FW–1 to about 13,000 ± 2,287 nmol g FW–1 within 4 h (Fig. 3B). PAL expression increased slightly in tissue fed with Phe when compared with that in petal limbs on intact plants, while PAL activity remained unchanged (Fig. 3C). In the case of the BSMT gene, the situation was slightly different. BSMT expression decreased after 2 h of feeding, while similar to PAL activity, BSMT activity was not altered, presumably due to protein stability (Fig. 3D). These results show that PAL and BSMT activities did not change in the time frame of the experiment in response to Phe feeding. For this analysis, samples (total RNA and crude extracts) were prepared from petal tissues fed with Phe for 30, 60, 120, and 240 min under dark conditions and compared with samples isolated from petal tissue harvested at the same time points directly from intact petunia plants grown under a reverse photoperiod resulting in dark conditions at these time points. Feeding experiments were performed when volatile emission was the highest and most constant to eliminate the effect of rhythmicity.
Floral volatiles were collected for 30, 60, 120, and 240 min under dark conditions from excised corolla limbs of 2-d-old petunia flowers continuously fed with 2H5-Phe and analyzed by gas chromatography-mass spectrometry (GC-MS) for pool sizes and isotope abundances of benzaldehyde, methylbenzoate, and phenylacetaldehyde emitted to the gas phase. Because of the nocturnal nature of emission of petunia scent components, plants were grown in growth chambers with a reversed photoperiod, and all experiments began at the same time of the day. After scent collections, petal tissues were extracted with either methanol and analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine the labeling and pool sizes of endogenous nonvolatile metabolites, including benzoic acid and its conjugates, or with hexane, to determine pool sizes and isotope abundances of endogenous intermediates and end products, including phenylacetaldehyde, phenylethanol, phenylethyl benzoate, eugenol, isoeugenol, benzaldehyde, benzyl alcohol, benzylbenzoate, and methylbenzoate. Figure 4 shows that isotopomers synthesized from supplied 2H5-Phe can be easily distinguished from unlabeled compounds based on mass spectra by mass spectrometry (MS). For all compounds except eugenol and isoeugenol, isotope abundance was determined as (2H5 x 100)/(2H5 + 2H0) (atom%). However, newly synthesized labeled eugenol and isoeugenol exhibit a shift of only +3 atomic mass units (amu) because the two hydroxylations of the caffeate moiety remove two deuterium atoms from the ring of the supplied 2H5-Phe. Thus, for eugenol and isoeugenol, isotope abundance was determined as (2H3 x 100)/(2 H3 + 2H0) (atom%; Fig. 4).
2H5-Phe was rapidly incorporated into benzaldehyde and phenylacetaldehyde, resulting in approximately 70% labeling for both compounds within 30 min after feeding that increased only slightly (by an additional 5%) during the next 3.5 h of feeding (Fig. 5A). Over a time course the pool of benzaldehyde was more extensively labeled than the benzoic acid pool, which was labeled more heavily than the methylbenzoate pool, suggesting that benzaldehyde is an intermediate in benzoic acid biosynthesis from Phe in petunia petals (Fig. 5A). To confirm the role of benzaldehyde as an intermediate in the pathway from Phe to benzoic acid, 2H5-benzaldehyde (37.5 µmol) was fed to petal tissue for 4 h with a simultaneous collection of volatiles. Supplied 2H5-benzaldehyde was converted to benzoic acid and methylbenzoate with the endogenous benzoic acid pool labeled to a greater extent than emitted methylbenzoate (72.4% and 36.3%, respectively; Fig. 5B). Phenylacetaldehyde was 100% unlabeled, indicating that it cannot be synthesized from benzaldehyde.
To determine whether formation of benzaldehyde from Phe occurs via CA, we conducted experiments with a specific inhibitor of PAL, 2-aminoindanephosphonate (AIP; Zon and Amrhein, 1992 ; Appert et al., 2003). In these experiments, excised corolla limbs were pretreated with AIP (25 µmol) for 40 min and then floral scent was collected for 4 h from limbs continuously fed with 2H5-Phe. Treatment with AIP almost completely eliminated emission of methylbenzoate, whereas emission of benzaldehyde decreased by 74% when compared with excised petals pretreated with water instead of AIP (Fig. 5C). AIP did not inhibit the emission of phenylacetaldehyde, and, in fact, it increased it by approximately 2-fold, suggesting that the formation of phenylacetaldehyde from Phe does not occur via CA and that its synthesis may be in competition with CA synthesis for Phe utilization.
Endogenous intermediates and end products in the benzenoid network were analyzed for pool sizes and isotope abundances after feeding corolla limbs with 2H5-Phe for 30, 60, 120, and 240 min under dark conditions. After hexane or methanol extraction, samples were analyzed by GC-MS or LC-MS, and experimentally obtained results are summarized in Figure 6B. These data were combined with pool sizes and isotope abundances of the volatile fraction and inhibitor studies and subjected to computer-assisted isotopic flux analysis (Stephanopoulos et al., 1998
In order to simultaneously account for the observed labeling patterns of benzoic acid (BA), endogenous and exogenous benzaldehyde (BAld), endogenous benzylbenzoate, and both the endogenous and exogenous pools of methylbenzoate (MB), both benzylbenzoate and benzaldehyde must be intermediates between Phe and benzoic acid. This indicates that both the CoA-dependent, -oxidative and CoA-independent, non--oxidative pathways are responsible for the biosynthesis of benzenoid compounds in petunia flowers. It was impossible to account for the labeling patterns of these metabolites and observed emission rates when only a single pathway was assumed to be metabolically active. The involvement of benzaldehyde as an intermediate in the biosynthesis of methylbenzoate from Phe was confirmed by the fact that deuterium-labeled benzaldehyde supplied to the petals was converted to benzoic acid and methylbenzoate (Fig. 5B).
The flux from CA
The large pool of benzylbenzoate (200 nmol g FW–1) accounted for the reduced labeling of benzoic acid, with both moieties of benzylbenzoate contributing to this dilution via different routes. The benzyl alcohol moiety reduced the labeling of benzoic acid indirectly via the dilution of benzaldehyde labeling. The experimentally obtained benzaldehyde labeling was consistent with the flux from benzyl alcohol to benzaldehyde acting as a reversible step (Fig. 6A). The benzoic acid moiety of benzylbenzoate directly contributed to the dilution of benzoic acid labeling. This moiety of benzylbenzoate is derived from benzoyl-CoA, which is synthesized primarily from the CoA-dependent,
To test the model-derived precursor-product relationships, 2H7-benzyl alcohol (37.5 µmol) was supplied to excised petunia petals for 4 h, and endogenous intermediates and end products in the benzenoid network were analyzed for pool sizes and isotope abundances. Supplied 2H7-benzyl alcohol led to the labeling of benzaldehyde (approximately 90%), methylbenzoate (approximately 42%), and both moieties of benzylbenzoate with the benzyl alcohol moiety more heavily labeled (approximately 56%) than the benzoic acid moiety (approximately 25%). These results confirmed that conversion of benzaldehyde to benzyl alcohol is reversible. The lower labeling of the benzyl alcohol moiety of benzylbenzoate relative to benzaldehyde labeling, each requiring one step with almost the same flux (v14 = 1.5 nmol min–1 g FW–1 and v17 = 1.4 nmol min–1 g FW–1), as was predicted by the model (Fig. 6A), is due to its dilution by the large pool size of unlabeled benzylbenzoate. The labeling of the benzoic acid moiety of benzylbenzoate is possible only if a flux from benzoic acid to benzoyl-CoA exists, confirming the model prediction of the reversibility of benzoyl-CoA Over a time course, LC-MS analyses not only revealed changes in endogenous pool sizes and isotope abundances of benzoic acid but also identified two unknown endogenous benzoic acid derivatives (C1 and C2), one of which is likely a glucoside of benzoic acid (Fig. 6A). At least one of these conjugates (C1) is metabolized back to benzoic acid, contributing to isotope dilution of the benzoic acid and methylbenzoate pools. We did not find any other labeled intermediates in LC-MS analyses, which could be due to our extraction procedure, pool sizes below our detection level, or channeling of these compounds. However, the model predicted the existence of other fates of benzoic acid, which were not yet identified. At the earliest sampling time, the emitted methylbenzoate was more highly labeled (18% at the 30-min time point) than would be expected from the experimentally obtained total internal methylbenzoate labeling (11%), eliminating the possibility of the existence of a single pool. It was necessary to invoke a large (75% of total), metabolically inactive (storage) pool of methylbenzoate, likely localized in the vacuole, in slow exchange (v29 = 0.05 nmol min–1 g FW–1) with a metabolically active pool, resulting in reduced labeling of the total extractable endogenous methylbenzoate (Fig. 6A).
Formation of phenylacetaldehyde from Phe probably occurs via a small pool of the intermediate phenylethylamine or phenylpyruvic acid (Fig. 1). The assumption that phenylacetaldehyde is the only precursor of phenylethanol led to the overestimation of the isotopic abundance of the latter, indicating that phenylacetaldehyde could only make a small contribution to phenylethanol labeling (Fig. 6A). A quantitative explanation of the labeling kinetics of phenylethanol suggests that the major flux to phenylethanol goes through a different route, possibly through phenylpyruvate and phenyllactic acid, as has been reported recently in rose flowers (Watanabe et al., 2002
The labeling kinetics of isoeugenol and eugenol (Fig. 6B) were consistent with synthesis from trans-cinnamate (via 4-coumarate and caffeic acid derivatives) in a 5:1 flux ratio, respectively. Because methyleugenol and methylisoeugenol were not found in the hexane extracts and were not emitted to the gas phase, and because eugenol and isoeugenol both behaved as metabolically inert end products, it can be assumed that flowers of petunia cv Mitchell lack the (iso)eugenol O-methyltransferase activity that was previously found in C. breweri (Wang et al., 1997
To isolate the gene(s) responsible for benzylbenzoate formation in petunia, we searched a recently generated expressed sequence tag (EST) collection (1,584 sequences) from a cDNA library constructed from mRNA isolated from petunia corolla limbs of buds to 2-d-old flowers for potential acyltransferases. This search revealed one EST clone of 1,637 nucleotides (1-1-F06) with 90% amino acid identity to a recently isolated BEBT from Nicotiana tabacum (D'Auria et al., 2002
To determine the enzymatic activity of the putative BEBT protein, the coding region of the gene was subcloned into the expression vector pET-11d, expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity using a weak anion-exchange DEAE-cellulose column followed by Mono-Q anion-exchange chromatography and by size-exclusion chromatography. The purified recombinant protein was used to evaluate its ability to catalyze the transfer of benzoyl and acetyl moieties to a large variety of potential substrate alcohols (Fig. 8) and to determine its general catalytic properties and kinetic parameters with the preferred substrates (Table I).
With acetyl-CoA, the highest activity of recombinant protein was found with benzyl alcohol as a substrate, and approximately 7- to 14-fold lower activities were detected with 3-hydroxy-benzyl alcohol, geraniol, and 2-phenylethanol (Fig. 8). Activities with the other tested substrates, including butanol, 1-octanol, 4-hydroxy-benzyl alcohol, 2-hexanol, cis-3-hexen-1-ol, and linalool, did not exceed 3% of the highest activity (Fig. 8). With benzoyl-CoA, the protein displayed a broad substrate preference, using efficiently a number of substrates, including benzyl alcohol, 3-hydroxy-benzyl alcohol, 2-phenylethanol, butanol, cis-3-hexen-1-ol, and 1-octanol (Fig. 8).
Kinetic characterization of the purified recombinant protein revealed that the apparent Km for acetyl-CoA is very similar to the value recently reported for C. breweri BEBT (682 µM and 818 µM for petunia and Clarkia proteins, respectively [D'Auria et al., 2002
Analysis of BPBT gene expression in leaves and different flower organs of 2-d-old petunia flowers by RNA gel-blot analysis using the BPBT coding region as a probe revealed BPBT mRNA transcripts predominantly in the limb of petunia corollas (Fig. 9A), the parts of the flower that were previously shown to be primarily responsible for scent production and emission in petunia (Kolosova et al., 2001
Benzenoid Network in Petunia Flowers
Benzenoid compounds are among the widespread fragrant components in the plant kingdom, where they contribute significantly to total floral scent output (Knudsen et al., 1993
Mathematical models can handle the large number of interacting variables of metabolic networks, assist in explaining the sometimes counter-intuitive behavior of metabolic networks in response to genetic or other perturbations, and provide information on the relative flux of carbon through a number of key reactions and intermediates (Bailey, 1998
Formation of benzenoid compounds from Phe requires the shortening of the side chain by a C2 unit that could occur via the CoA-dependent,
The metabolic flux model developed in this study, combined with the PAL inhibition data, revealed that the formation of phenylpropanoid-related compounds (phenylacetaldehyde, phenylethanol, and the phenylethanol moiety of phenylethyl benzoate) from Phe does not occur via CA (Figs. 5C and 6A). Modeling also revealed that both the CoA-dependent,
Modeling of 2H5-Phe labeling data indicated that benzylbenzoate is one of the intermediates between L-Phe and benzoic acid. Benzylbenzoate is formed from benzyl alcohol and benzoyl-CoA in a reaction catalyzed by a benzoyl transferase, activity for which was found in petunia petal tissue (Fig. 10A). The activity of this enzyme over flower development showed a typical profile of scent biosynthetic enzymes, where high enzymatic activities were found in old flowers without concomitant emission of corresponding products (Dudareva and Pichersky, 2000 Using a functional genomic approach, we have isolated a BPBT gene from a petunia petal-specific EST collection that encodes a protein capable of synthesizing benzylbenzoate (Fig. 7). This gene was expressed almost exclusively in the limbs of petunia corollas (Fig. 9A), and its expression was developmentally and rhythmically regulated (Fig. 9, B and C). During a daily light/dark cycle, its expression peaked around 3 PM, several hours before maximum benzylbenzoate accumulation (Fig. 10C). This earlier expression of the BPBT gene is probably required for building a large pool of benzylbenzoate (200 nmol g FW–1), which contributes to methylbenzoate emission at night via benzoic acid. Biochemical characterization of the BPBT protein showed that it can use a broad range of alcohols in the presence of benzoyl-CoA, one of which is 2-phenylethanol (Fig. 8). Benzoylation of phenylethanol results in the formation of phenylethyl benzoate, a phenylpropanoid-related compound found in petunia petal tissue. The BPBT catalytic efficiency with phenylethanol was close to that with benzyl alcohol (Table I), suggesting that this enzyme is most likely responsible for the formation of both phenylethyl benzoate and benzylbenzoate in vivo. However, the internal pool of benzylbenzoate (200 nmol g FW–1) was 14 times larger than that of phenylethyl benzoate (14 nmol g FW–1) due to the larger amount of metabolically active benzyl alcohol (2 nmol g FW–1) relative to phenylethanol (0.1 nmol g FW–1) available to the enzyme.
The model developed in this study indicated that benzoyl-CoA, one of the BPBT substrates for formation of both phenylethyl benzoate and benzylbenzoate, is synthesized via the CoA-dependent,
Although our results do not show any existence of channeling of metabolic intermediates in our established steps of the benzenoid branchway, the possibility exists that channeling of metabolic intermediates occurs through multienzyme complexes early in the pathway (before benzaldehyde), which may result in little or no release of the label into the bulk extractable pools (Hrazdina, 1992
Our model did not address the regulatory architecture of the benzenoid network (Stephanopoulos and Vallino, 1991
Plant Material, Chemicals, and Radiochemicals
Petunia (Petunia hybrida) cv Mitchell (Ball Seed, West Chicago, IL) were grown under standard greenhouse conditions as described previously (Kolosova et al., 2001
Deuterium-labeled Phe (L-Phe-ring-2H5) and benzyl alcohol (2H7-benzyl alcohol) were purchased from Cambridge Isotope Laboratories (Andover, MA), deuterium-labeled benzaldehyde (benzaldehyde-2H6) was from Isotec (Miamisburg, OH), L-Phe was from Sigma (St. Louis), and radiolabeled Phe, U-[14C]Phe (425 mCi/mmol), was from American Radiolabeled Chemicals (St. Louis). Radiolabeled [acetyl-1-14C]CoA (55 mCi/mmol) was purchased from MP Biomedicals (Irvine, CA) and S-[methyl-14C]adenosyl-L-Met (52.7 mCi/mmol) was from Perkin-Elmer Life Sciences (Boston). [7-14C]benzoyl-CoA (53 mCi/mmol) was enzymatically synthesized from [7-14C]benzoic acid (Beuerle and Pichersky, 2002a
Stable isotope labeling was conducted in a glass container by placing the cut surface of excised corollas of 2-d-old petunia flowers on moist filter paper supplied with 2H5-Phe (total 10 corollas of 0.2 g each per experiment). A constant stream of air flowing at 450 mL min–1 was drawn through the container with a vacuum pump. The air exiting the sample-filled container was passed through an adsorbent trap consisting of a 120- x 7-mm glass tube containing 100 mg of polymer Porapak Type Q (80/100 mesh; Alltech, Deerfield, IL) held in place with plugs of silanized glass wool. An additional adsorbent trap of identical construction was placed at the inlet of the container to purify incoming air. Trials with two such traps connected in series at the container exit indicated that there was no detectable breakthrough (i.e. no loss of floral volatiles from the first trap due to overloading, even when collections were carried out for periods of up to 24 h). Since petunia cv Mitchell is a nocturnally emitting plant (Kolosova et al., 2001
Trapped volatiles were eluted from the trap with hexane, and the amount and isotope abundance of benzenoids emitted to the gas phase were analyzed by GC-MS with a FinniganMAT GCQ instrument (Thermoquest, San Jose, CA; injector temperature 230°C, injector volume 1 µL, and split ratio 50:1) using a DB-1 nonpolar capillary column (30 m x 0.25 mm [i.d.]; film thickness 0.25 µm). Ionization energy was set at 70 eV. Column temperature was held at 50°C for 1 min and then heated to 240°C at 10°C min–1. Mass spectra were obtained in scan mode scanning across 41 to 400 amu. Components were first identified from a computer database containing several thousand mass spectra and were then confirmed by comparing retention times and mass spectra with those of authentic standards. Quantification was based on flame ionization detector peak areas and the internal standards, toluene and napthalene. Single ion monitoring was generally used for the measurements of mass isotopomers. All newly synthesized labeled benzenoid compounds exhibit a mass shift by 5 amu, except eugenol and isoeugenol, which exhibit a shift of only by 3 amu. Two moieties of benzylbenzoate corresponding to the benzoic acid and benzyl alcohol moieties of the molecule were labeled to different extents, and their labeling was determined separately. The percentage of labeling was determined as the intensity of the shifted representative molecular ion divided by the sum of intensities for unshifted and shifted representative molecular ions. To determine pool sizes and isotope abundance of endogenous pools of intermediates and end products, including phenylacetaldehyde, phenylethanol, eugenol, isoeugenol, benzaldehyde, benzylbenzoate, and methylbenzoate, corolla tissues after feeding were extracted with hexane. Tissue was ground in liquid nitrogen and hexane was added to 4 mL/g of tissue. Tissue was extracted on an Orbit shaker at 170 rpm for 1 h and extract was centrifuged at 10,000 rpm for 10 min, followed by filtration through a 25-mL syringe with a 0.2 µM sterile nylon filter. Filtered extract was evaporated down to 160 µL, and samples were then analyzed by GC-MS as mentioned above. All samples were corrected for recovery as determined using internally spiked samples. To determine the labeling of endogenous nonvolatile metabolites, including benzoic acid and its conjugates, labeled corolla tissue was extracted with methanol and analyzed by LC-MS.
To determine the internal pool sizes of Phe, corolla tissues were extracted with methanol followed by phase separation with chloroform and water (10 mL of methanol:5 mL of chloroform:6 mL of water; Rhodes et al., 1987
After scent collection, petal tissue was extracted with methanol and the isotope abundance of endogenous nonvolatile intermediates was analyzed by LC-MS. Prior to entrance into the mass spectrometer, extracted compounds were separated using a Supelco Discovery HS C18 column (15 cm x 2.1 mm i.d.) attached to a Waters 2690 separations module (Milford, MA) with attached column oven. Compound elution was monitored at 210 and 280 nm with a Waters 996 UV/Visible photodiode array detector. Complete baseline separation of all phenylpropanoid compounds was achieved at a flow rate of 0.25 mL min–1 with the column incubated at constant temperature of 40°C. Solvent A was 0.05% formic acid in water; solvent B was 100% acetonitrile. The column was pre-equilibrated with 5% solvent B in solvent A. After injection of up to 25 µL of aqueous sample, the column was washed with 0.5 mL of pre-equilibration solvent. Compounds were eluted from the column with a linear gradient from 5% to 66% solvent B over 13.75 mL. The column was then washed by increasing solvent B to 100% (linear gradient in 0.75 mL) and holding at 100% solvent B for 0.75 mL. The column was then re-equilibrated by returning the column to 5% solvent B (over 0.75 mL) followed by a 2.5 mL wash with this solvent. Total run time was 70 min. In-line mass spectrometry (LC-MS) was performed on HPLC eluents using a Micromass Quattro LCZ triple quadruple mass spectrometer (Micromass, Beverly, MA). Flow splitting (10:1) after the Waters 996 photodiode array detector resulted in an inlet flow rate into the mass spectrometer electrospray source (ESI Z-Spray; Micromass) of approximately 20 to 25 µL min–1. This dramatically enhanced the signal when compared to nonsplit samples. Ionization of target molecules in negative ion mode was achieved with a capillary voltage of 3.0 kV and a cone voltage of 30 V. The desolvation and cone gases were set at 430 and 60 L h–1, respectively, and the desolvation and source temperatures were 250°C and 120°C, respectively. Mass detection was performed in negative ion scanning mode, at 450 amu s–1, with 0.1-s interscan delay. All other electrospray source and instrument parameters were set as recommended by the instrument manufacturer. Data analysis was performed using MassLynx computer software (Micromass).
The computer model used was similar to the metabolic flux analysis models described by Kocsis et al. (1998)
For all metabolites, the rate of change of the concentration of metabolite M is taken as
During short time intervals (0.125-min iterations), material of the current isotopic abundance was drawn from one pool to another at specified rates, new isotopic abundances and pool sizes were computed, and isotopic abundance in each pool was plotted (superimposed on observed data) as a function of time. The model was primarily constrained by observed rates of emission of methylbenzoate, benzaldehyde, and phenylacetaldehyde to the gas phase (as determined by GC and GC-MS), and observed pool sizes of benzoic acid and its conjugates (C1 and C2; as determined by LC-MS). Other flux rates and pool sizes were progressively adjusted until a close match between observed and simulated isotopic abundance was obtained, as judged graphically or by computing absolute deviations between observed and simulated values. Further details on the development of the type of metabolic models used here are given at http://www.hort.purdue.edu/cfpesp/models/models.htm.
A directional cDNA library was constructed from mRNA isolated from petunia petal tissues of buds 1 d before opening to 2-d-old flowers using ZAP express cDNA synthesis kit (Stratagene, La Jolla, CA). The titer of the unamplified library was 2.8 x 106 plaque forming units. This primary library was amplified, and the amplified library had a titer of 7 x 109 plaque forming units. Mass excision of pBluescript phagemids (Stratagene) from the amplified library resulted in a stock, which was used for plating and random colony picking for DNA sequencing. A total of 1,584 cDNAs were automatically isolated and sequenced from their 5' end using T3 primer. After vector removal, the resulting petunia ESTs were compared with GenBank and dbEST using the BLASTX and TBLASTX search algorithms.
The coding region of petunia BPBT was amplified by PCR using the forward primer 5'-CTTCCATGGATTCAAAGCAATCATCAGA-3', which introduced an NcoI site at the initiating ATG codon in combination with the reverse primer 5'-CCTAGCTCATAGGGCAGGTGTGATAAAGGC-3', which introduced a BamHI site downstream of the stop codon and subcloned into the expression vector pET 11d (Novagen, Madison, WI). Sequencing revealed that no errors had been introduced during PCR amplifications.
For functional expression, E. coli BL834 (DE3) cells were transformed with the resulting recombinant plasmid and pET vector without an insert as control, and grown in Luria-Bertani medium with 100 µg/mL ampicilin at 37°C. When the culture density reached OD600 of 0.5, the expression of BEBT cDNA was induced by addition of isopropyl-1-thio- First, BPBT protein was loaded onto a DEAE-cellulose column (18 mL of DE53; Whatman, Clifton, NJ) preequilibrated with buffer A at a flow rate of about 1 mL/min. After washing off unabsorbed material from the column with 60 mL of buffer A, BPBT was eluted with a linear gradient (120 mL) from 0 to 500 mM KCl in buffer A. Fractions (3 mL) were collected and assayed for BPBT activity with benzyl alcohol as a substrate. Fractions with the highest BPBT activity in the 140 to 225 mM KCl range were pooled (total of 18 mL), dialyzed against buffer A, and subjected to ion-exchange chromatography on a Mono-Q column (gel bed volume 1 mL) (Amersham Biosciences, Piscataway, NJ) using the FPLC system. BPBT protein was eluted from the column at 80 to 160 mM KCl using a 15-mL linear (0–400 mM) gradient of KCl in buffer A at flow rate of 0.5 mL/min. Fractions with highest BPBT activity in the 75 to 165 mM KCl range (total of 4 mL) were pooled and subjected to size-exclusion chromatography (Superdex 200; 16 mm x 60 cm). Fractions of 2 mL were collected at a flow rate of 1 mL/min and analyzed for BPBT activity. Fractions with highest BPBT activity were examined by SDS-PAGE gel electrophoresis followed by Coomassie Brilliant Blue staining of the gel.
For kinetic analysis, an appropriate enzyme concentration was chosen so that the reaction velocity was proportional to the enzyme concentration and was linear with respect to incubation time for at least 30 min. Protein concentrations were determined by the Bradford method (Bradford, 1976
Crude protein extracts were made from corolla limbs of petunia flowers as described previously (Dudareva et al., 2000 Verification of products produced by BPBT in benzoylation and acylation reactions was achieved by performing enzyme assays as described above only using nonradioactive benzoyl-CoA and acetyl-CoA instead of radioactive ones and analyzing the products by GC-MS.
Total RNA was isolated and analyzed as previously described (Dudareva et al., 1996 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY611496.
We thank Dr. Yoko Iijima and Dr. Eran Pichersky for their generous gift of [7-14C]benzoyl-CoA, and Dr. Eran Pichersky for sharing with us 2-aminoindane-phosphonate. We also thank Dr. John C. D'Auria for his helpful discussions and Dr. Stanislav Zakharov for his help with the gel filtration chromatography. Received April 28, 2004; returned for revision June 23, 2004; accepted June 24, 2004.
1 This work was supported by the U.S. Department of Agriculture National Research Initiative (grant no. 2003–35318–13619) and by the Fred Gloeckner Foundation (grant to N.D.). This paper is contribution Number 17397 from the Purdue University Agricultural Experimental Station.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.045468. * Corresponding author; e-mail dudareva{at}hort.purdue.edu; fax 765–494–0391.
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benzaldehyde 
-amino-n-butyrate as an internal standard, as previously described (Rhodes et al., 1987
