Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration.

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ß-oxidative or nonoxidative pathways. The first step in the ß-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 ß-oxidative pathway.

Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids.

RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.

l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta.

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.

MicroRNA modulation in complex regional pain syndrome.

BACKGROUND: Aberrant expression of small noncoding RNAs called microRNAs (miRNAs) is a common feature of several human diseases. The objective of the study was to identify miRNA modulation in patients with complex regional pain syndrome (CRPS) a chronic pain condition resulting from dysfunction in the central and/or peripheral nervous systems. Due to a multitude of inciting pathologies, symptoms and treatment conditions, the CRPS patient population is very heterogeneous. Our goal was to identify differentially expressed miRNAs in blood and explore their utility in patient stratification. METHODS: We profiled miRNAs in whole blood from 41 patients with CRPS and 20 controls using TaqMan low density array cards. Since neurogenic inflammation is known to play a significant role in CRPS we measured inflammatory markers including chemokines, cytokines, and their soluble receptors in blood from the same individuals. Correlation analyses were performed for miRNAs, inflammatory markers and other parameters including disease symptoms, medication, and comorbid conditions. RESULTS: Three different groups emerged from miRNA profiling. One group was comprised of 60% of CRPS patients and contained no control subjects. miRNA profiles from the remaining patients were interspersed among control samples in the other two groups. We identified differential expression of 18 miRNAs in CRPS patients. Analysis of inflammatory markers showed that vascular endothelial growth factor (VEGF), interleukin1 receptor antagonist (IL1Ra) and monocyte chemotactic protein-1 (MCP1) were significantly elevated in CRPS patients. VEGF and IL1Ra showed significant correlation with the patients reported pain levels. Analysis of the patients who were clustered according to their miRNA profile revealed correlations that were not significant in the total patient population. Correlation analysis of miRNAs detected in blood with additional parameters identified miRNAs associated with comorbidities such as headache, thyroid disorder and use of narcotics and antiepileptic drugs. CONCLUSIONS: miRNA profiles can be useful in patient stratification and have utility as potential biomarkers for pain. Differentially expressed miRNAs can provide molecular insights into gene regulation and could lead to new therapeutic intervention strategies for CRPS.

Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection.

A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03 M potassium dihydrogen phosphate (pH 6.5)-acetonitrile 65:35 (v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 274 and 370 nm, respectively. Calibration plots were linear over the concentration range 10-1,000 ng mL(-1) in human plasma, and the lower limit of quantification was 10 ng mL(-1). Accuracy was between 0.4 and 6.4% and precision was between 3.1 and 7.5%. Amisulpride was sufficiently stable through three freeze-thaw cycles, during storage for 6 h at room temperature, and for 2 months at -22 °C. The method is suitable for the analysis of clinical samples from pharmacokinetic studies.

The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene.

Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the artificial hybrid Petunia hybrida, a cross between P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subsp. parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent-synthesizing and scent-emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide-like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame-shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by an as yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNA interference also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.

The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages.

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.

Circulating microRNA Signatures in Rodent Models of Pain.

MicroRNAs (miRNAs) remain stable in circulation and have been identified as potential biomarkers for a variety of conditions. We report miRNA changes in blood from multiple rodent models of pain, including spinal nerve ligation and spared nerve injury models of neuropathic pain; a complete Freund's adjuvant (CFA) model of inflammatory pain; and a chemotherapy-induced model of pain using the histone deacetylase inhibitor JNJ-26481585. The effect of celecoxib, a cyclooxygenase-2-selective nonsteroidal anti-inflammatory drug, was investigated in the CFA model as proof of principle for assessing the utility of circulating miRNAs as biomarkers in determining treatment response. Each study resulted in a unique miRNA expression profile. Despite differences in miRNAs identified from various models, computational target prediction and functional enrichment have identified biological pathways common among different models. The Wnt signaling pathway was affected in all models, suggesting a crucial role for this pathway in the pathogenesis of pain. Our studies demonstrate the utility of circulating miRNAs as pain biomarkers and suggest the potential for rigorous forward and reverse translational approaches. Evaluating alterations in miRNA fingerprints under different pain conditions and after administering therapeutic agents may be beneficial in evaluating clinical trial outcomes, predicting treatment response, and developing correlational outcomes between preclinical and human studies.

Regulation of actomyosin contractility by PI3K in sensory axons.

Phosphatidylinositol 3-kinase (PI3K) activity is known to be required for the extension of embryonic sensory axons. Inhibition of PI3K has also been shown to mediate axon retraction and growth cone collapse in response to semaphorin 3A. However, the effects of inhibiting PI3K on the neuronal cytoskeleton are not well characterized. We have previously reported that semaphorin 3A-induced axon retraction involves activation of myosin II, the formation of an intra-axonal F-actin bundle cytoskeleton, and blocks the formation of F-actin patches that serve as precursors to filopodial formation in axons. We now report that inhibition of PI3K results in activation of myosin II in axons. Inhibition of myosin II activity, or its upstream regulatory kinase RhoA-kinase, blocked axon retraction induced by inhibition of PI3K. In addition, inhibition of PI3K also induced intra-axonal F-actin bundles, which likely serve as a substratum for myosin II-based force generation during axon retraction. In axons, filopodia are formed from axonal F-actin patch precursors. Analysis of axonal F-actin patch formation in eYFP-actin expressing neurons revealed that inhibition of PI3K blocked formation of axonal F-actin patches, and thus filopodial formation. These data provide insights into the regulation of the neuronal cytoskeleton by PI3K and are consistent with the notion that decreased levels of PI3K activity mediate axon retraction and growth cone collapse in response to semaphorin 3A.

Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a beta-oxidative or non-beta-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between L-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester.

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.

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation.

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 (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.

The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers.

Terpenoids, the largest class of plant secondary metabolites, play essential roles in both plant and human life. In higher plants, the five-carbon building blocks of all terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, are derived from two independent pathways localized in different cellular compartments. The methylerythritol phosphate (MEP or nonmevalonate) pathway, localized in the plastids, is thought to provide IPP and dimethylallyl diphosphate for hemiterpene, monoterpene, and diterpene biosynthesis, whereas the cytosol-localized mevalonate pathway provides C5 units for sesquiterpene biosynthesis. Stable isotope-labeled, pathway-specific precursors (1-deoxy-[5,5-2H2]-D-xylulose and [2,2-2H2]-mevalolactone) were supplied to cut snapdragon flowers, which emit both monoterpenes and the sesquiterpene, nerolidol. We show that only one of the two pathways, the plastid-localized MEP pathway, is active in the formation of volatile terpenes. The MEP pathway provides IPP precursors for both plastidial monoterpene and cytosolic sesquiterpene biosynthesis in the epidermis of snapdragon petals. The trafficking of IPP occurs unidirectionally from the plastids to cytosol. The MEP pathway operates in a rhythmic manner controlled by the circadian clock, which determines the rhythmicity of terpenoid emission.

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 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.

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants.

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.

Understanding in vivo benzenoid metabolism in petunia petal tissue.

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 C(2) unit. Deuterium-labeled Phe ((2)H(5)-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, beta-oxidative and CoA-independent, non-beta-oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non-beta-oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, beta-oxidative pathway. Modeling of (2)H(5)-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.