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, β-oxidative and CoA-independent, non-β-oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non-β-oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, β-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.

Snapdragon flowers emit two monoterpene olefins, myrcene and ( E )-␤ -ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases ( ama1e20 and ama0c15 ) and an ( E )-␤ -ocimene synthase ( ama0a23 ). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. ( E )-␤ -Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly ( E )-␤ -ocimene (97% of total monoterpene olefin product) with small amounts of ( Z )-␤ -ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx 8 W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S -linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (ϳ65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent K m of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO 2 , ammonia, and hydrogen peroxide in stoichiometric amounts.

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.floral scent ͉ phenylpropanoids ͉ phenylpropenes ͉ plant volatiles ͉ secondary compounds

The precursor of all monoterpenes is the C 10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C 5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.

The molecular mechanisms responsible for postpollination changes in floral scent emission were investigated in snapdragon cv Maryland True Pink and petunia cv Mitchell flowers using a volatile ester, methylbenzoate, one of the major scent compounds emitted by these flowers, as an example. In both species, a 70 to 75% pollination-induced decrease in methylbenzoate emission begins only after pollen tubes reach the ovary, a process that takes between 35 and 40 h in snapdragon and ‫ف‬ 32 h in petunia. This postpollination decrease in emission is not triggered by pollen deposition on the stigma. Petunia and snapdragon both synthesize methylbenzoate from benzoic acid and S -adenosyl-L -methionine (SAM); however, they use different mechanisms to downregulate its production after pollination. In petunia, expression of the gene responsible for methylbenzoate synthesis is suppressed by ethylene. In snapdragon, the decrease in methylbenzoate emission is the result of a decrease in both S -adenosyl-L -methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and the ratio of SAM to S -adenosyl-L -homocysteine ("methylation index") after pollination, although the BAMT gene also is sensitive to ethylene.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the b-oxidative or nonoxidative pathways. The first step in the b-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the b-oxidative pathway.

Emission of methyl benzoate, one of the most abundant scent compounds of bee-pollinated snapdragon flowers, occurs in a rhythmic manner, with maximum emission during the day, and coincides with the foraging activity of bumblebees. Rhythmic emission of methyl benzoate displays a "free-running" cycle in the absence of environmental cues (in continuous dark or continuous light), indicating the circadian nature of diurnal rhythmicity. Methyl benzoate is produced in upper and lower snapdragon petal lobes by enzymatic methylation of benzoic acid in the reaction catalyzed by S -adenosyl-L -methionine:benzoic acid carboxyl methyltransferase (BAMT). When a detailed time-course analysis of BAMT activity in upper and lower petal lobes during a 48-hr period was performed, high BAMT activity was found at night as well as in continuous darkness, indicating that the BAMT activity is not an oscillation-determining factor. Analysis of the level of benzoic acid during a 24-hr period revealed oscillations in the amount of benzoic acid during the daily light/dark cycle that were retained in continuous darkness. These data clearly show that the total amount of substrate (benzoic acid) in the cell is involved in the regulation of the rhythmic emission of methyl benzoate. Our results also suggest that similar molecular mechanisms are involved in the regulation of methyl benzoate production in diurnally (snapdragon) and nocturnally (tobacco and petunia) emitting plants. INTRODUCTIONFlowers of many plant species attract pollinators by producing different complex mixtures of volatile compounds that give each species a unique and characteristic fragrance. Such volatile compounds emitted from flowers may function as both long-distance and short-distance attractants and play a prominent role in the localization and selection of flowers by insects, especially in moth-pollinated flowers, which are detected and visited at night (Dobson, 1994). Plants need to, and often do, vary the floral scent they emit during the life span of the flower, both in total output and in specific composition. These changes occur in relation to flower age (Tollsten, 1993;Pichersky et al., 1994;Wang et al., 1997; Dudareva et al., 1998Dudareva et al., , 2000, pollination status (Tollsten, 1993; Schiestl et al., 1997), environmental conditions (Jakobsen and Olsen, 1994), and diurnal endogenous rhythms (summarized by Dudareva et al., 1999). In addition to reduced costs for scent production, these intraspecific changes in floral fragrances also can influence the activity of flower visitors. For example, a quantitative and/or qualitative change of floral bouquets after pollination might lead to a lower attractiveness of these flowers and help to direct pollinators to the unpollinated flowers, thereby maximizing the reproductive success of the plant.It is an evolutionary advantage that plants have scent output at maximal levels only when potential pollinators are active. Plants that are pollinated by insects with maximum activity during the day (e.g., bees) show a diurnal...