Plant Physiol. (1998) 118: 209-218

Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway¹

Michel Schalk, Francisco Cabello-Hurtado, Marie-Agnès Pierrel, Rossitza Atanossova, Patrick Saindrenan, and Danièle Werck-Reichhart^*

Département d'Enzymologie Cellulaire et Moléculaire (M.S., F.C.-H., D.W.-R.), and Département de Phytopathologie (M.-A.P., R.A., P.S.), Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique Unité Propre de Recherche 406, 28 rue Goethe, 67000 Strasbourg, France

	ABSTRACT

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Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (K_I = 17 µM, k_inact = 0.064 min¹), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

	INTRODUCTION

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The phenylpropanoid metabolism is a plant-specific pathway leading to compounds of extremely diverse structure and function (Dixon and Paiva, 1995; Werck-Reichhart, 1995). It is involved in the formation of quantitatively major biopolymers such as lignin and suberin, but also in the biosynthesis of signaling molecules such as salicylic acid and isoflavonoids in flower pigments, UV protectants such as anthocyanins, flavonoids, and coumarins, and several classes of phytoalexins. The upstream part of the phenylpropanoid metabolism, which branches from the shikimate pathway at the level of L-Phe, consists of a core of three enzymatic steps leading to 4-coumaroyl CoA (Fig. 1). This set of three reactions, often called the general phenylpropanoid pathway, controls the flux of metabolites toward all families of compounds derived from the C6-C3 skeleton of Phe. Compounds with a C6-C1 structure are not strictly phenylpropanoids, but also derive from L-Phe. They originate from the core pathway intermediates cinnamic acid or 4-coumaric acid (Yalpani et al., 1993). Molecules with a C6-C1 backbone include benzoic and salicylic acids and economically important compounds such as vanillin.

View larger version (15K): [in this window] [in a new window]   Figure 1. Branching and inhibitors of the phenylpropanoid pathway. AOPP, -Amino--phenylpropionic acid; MDCA, methylenedioxycinnamic acid.

The second step in the core phenylpropanoid pathway is the hydroxylation of trans-cinnamic acid to 4-coumaric acid. The reaction is catalyzed by C4H, a member of the superfamily of Cyt P450 heme-thiolate proteins. P450s are monooxygenases that catalyze the oxidation of a remarkably broad range of endogenous and exogenous chemicals in all organisms. In plants they play important roles in biosynthetic pathways, including those of sterols, isoprenoids, alkaloids, oxygenated fatty acids, and phenylpropanoids (Bolwell et al., 1994; Durst and O'Keefe, 1995; Schuler, 1996). They are also involved in the metabolism and sometimes in the activation of many herbicides, insecticides, and other xenobiotics. CYP73A1 is a C4H from Jerusalem artichoke (Helianthus tuberosus). Its coding sequence was isolated from tuber tissues (Teutsch et al., 1993) and expressed in yeast (Urban et al., 1994). The yeast-expressed enzyme is catalytically active and capable of hydroxylating cinnamic acid with a very high efficiency (k_cat ranging from 100 to 400 min¹).

The high activity of the recombinant enzyme led us to investigate its activity toward other potential substrates, natural plant components, and xenobiotics. Several exogenous molecules were thus found to be substrates of CYP73A1 (Pierrel et al., 1994; Schalk et al., 1997). The efficiency of the metabolism of the xenobiotic molecules largely relied on their structural analogy to the natural substrate. A systematic structure-activity study recently led to the characterization of some good alternative substrates and high-affinity inhibitors of the enzyme, and of the structural requirements for an efficient binding into the active site of CYP73A1 (Schalk et al., 1997). The ideal ligand of C4H was thus defined as a rigid hydrophobic backbone of the size of a bicyclic aromatic structure (e.g. naphthalene), bearing one or several small negatively charged substituent(s) centered around carbon 2 of the naphthalene ring, the prototype alternative substrate being 2-naphthoic acid (Fig. 2).

View larger version (8K): [in this window] [in a new window]   Figure 2. Structural analogy between substrates of CYP73A1.

PA is a natural molecule extracted from the bark of the Paracoto tree that roughly fulfills all of these requirements. PA contains a MDP function at a position suitable for oxidative attack by CYP73A1. Many compounds with MDP function have been shown to inhibit mammalian or insect P450 enzymes both in vitro and in vivo (Franklin, 1977; Wilkinson et al., 1984; Ortiz de Montellano and Correia, 1995). They were shown to act as mechanism-based inactivators and to require P450-catalyzed metabolism to generate a MI forming a stable complex with the enzyme (Franklin, 1971). Available data suggest that the MI is likely a carbene that binds as the sixth coordinant to the heme iron (Mansuy et al., 1979).

We have tested PA inhibition of recombinant CYP73A1 and show that it behaves as a very potent, mechanism-based inhibitor of C4H. It is effective in vitro on the recombinant enzyme, being far more efficient than other MDP compounds. It is apparently selective for C4H. Assays performed in vivo on tobacco (Nicotiana tabacum) leaves and cell cultures indicate that it can be used to inactivate C4H and to block the input of precursors into the main C6-C3 pathway. To our knowledge, it is the first selective and quasi-irreversible inhibitor of the C4H so far described.

	MATERIALS AND METHODS

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Chemicals

Yeast and Plant Microsomes

Mat

Microsomes were prepared from Jerusalem artichoke (Helianthus tuberosus L. var. Blanc commun) tuber tissues that were sliced, aged, and treated with aminopyrine, MnCl₂, or CuCl₂ as described previously (Werck-Reichhart et al., 1990; Cabello-Hurtado et al., 1998b).

Spectrophotometric Measurements

Enzymatic Assays

1

Enzyme Inactivation

1

View larger version (26K): [in this window] [in a new window]   Figure 3. Binding of PA to CYP73A1. Difference spectra were recorded in 0.1 M sodium phosphate, pH 7.4, containing CYP73A1 (0.12 µM) in microsomes from transformed W(R) yeast. Solid lines correspond to the difference spectra obtained after addition of increasing amounts (5, 10, 30, 80, 180, and 380 µM) of PA to the sample cuvette (curves a-f). An equal volume of buffer was added to the reference. The broken line is the difference spectrum obtained under the same conditions after addition of 100 µM cinnamic acid. Inset, Double-reciprocal plot of A412-430 versus PA concentration.

Treatments of Leaf Tissues and Tobacco (Nicotiana tabacum L. cv Samsun, NN) Cell Suspension

The tobacco BY-2 cell suspension was maintained as described by Nagata et al. (1992). Synchronization of the cells was achieved according to the method of Reichheld et al. (1995) with 3 mg mL¹ aphidicolin and 1.5 mg mL¹ propyzamide. Elicitor treatment of cell-suspension cultures was performed 48 h after aphidicolin treatment with 50 nM -megaspermin, an elicitin protein purified from culture filtrates of Phytophthora megasperma (Baillieul et al., 1995). PA (100 µM) was added at the same time as megaspermin. Cells were harvested by vacuum filtration 13 h after treatment, and frozen in liquid nitrogen.

Analytical Procedures

	RESULTS

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Binding of PA to CYP73A1

Binding of trans-cinnamic acid to CYP73A1 results in a typical type I spectrum (Fig. 3). Addition of PA to a microsomal suspension of W(R) yeast expressing CYP73A1 also induced a shift in absorbance that clearly resulted from its binding in the P450 heme pocket (Fig. 3). The oxidized and PA-bound versus oxidized difference spectrum presented a peak at about 412 nm and a trough around 430 nm. The A₄₁₂₄₃₀ increased with PA concentration and saturation was obtained at a concentration of about 400 µM. The apparent K_s calculated from the double-reciprocal plot of A₄₁₂₄₃₀ versus substrate concentration was 45 ± 11 µM. The PA-induced spectrum was reminiscent of but not identical to the spectrum induced by cinnamic acid. The shift of the Soret maximum to 412 nm in the CYP73A1-PA complex possibly indicates a PA-induced alteration in the heme-protein structure or a direct interaction of PA with the iron-porphyrin system. The small amplitude of the absorbance change also suggests that the solvent was not completely displaced from the immediate heme environment.

Formation of a CYP73A1-MI Complex

View larger version (24K): [in this window] [in a new window]   Figure 4. Formation of a complex between the CYP73A1 and PA metabolite. CYP73A1 (0.12 µM) in transformed yeast microsomes was incubated at 30°C in 0.1 M sodium phosphate buffer, pH 7.4, containing 500 µM NADPH. PA was added to the sample cuvette at a final concentration of 30 µM. Difference spectra were recorded 1, 2, 3, 4, 5, 7, and 10 min after adding PA (curves a-g).

The increase in A₄₂₇₄₉₀ was recorded as a function of time for various concentrations of PA (Fig. 5). It displayed saturation kinetics as a function of both time and PA concentration. After 15 to 30 min of incubation, the amplitude of A₄₂₇₄₉₀ started to decline slowly. This was likely the result of a reoxidation of CYP73A1 (Franklin, 1971), since both formation and stability of the complex absorbing at 427 nm were enhanced by reduction of heme iron. The addition of sodium dithionite to both cuvettes after completion of the reaction with NADPH resulted in a slight increase in A₄₂₇ and in a complete disappearance of the peak at A₄₅₅. After complete reduction, the spectrum remained quite stable. A similar stabilization of the P450 complex by subsequent addition of sodium dithionite was observed with several MDP compounds (Hodgson et al., 1973; Yu et al., 1980).

View larger version (21K): [in this window] [in a new window]   Figure 5. Time- and inhibitor-concentration-dependence of the formation of the complex between CYP73A1 and PA. Formation of the complex was measured as described in Figure 4. Time-dependent appearance of the 427-nm peak was recorded for PA concentrations of 2 (), 5 (- - -), 15 ( · - · ), 30 (- - -), and 50 µM ( ·  ·  · - ·  ·  · ).

The 427-nm-absorbing species was not formed with sodium dithionite alone. Metabolic activation of PA is thus required, and the type III spectrum does not result from the direct binding of PA to reduced P450. These data strongly suggest that the absorbance changes observed in the presence of PA and NADPH actually reflect the formation of a P450-bound MI.

Reversibility of the P450-MI Complex

Addition of CO to the sample cuvette after stabilization of the complex with sodium dithionite did not shift the absorption to 450 nm. The P450-MI complex was thus not dissociated by CO (not shown), in agreement with previous reports (Hodgson et al., 1973).

Kinetics of CYP73A1 Inactivation by PA

1

View larger version (29K): [in this window] [in a new window]   Figure 6. Time- and concentration-dependent inactivation of CYP73A1 by PA. CYP73A1 in W(R) yeast microsomes was preincubated at 30°C in 0.1 M sodium phosphate, pH 7.4, with 50 µM NADPH, 1 mM Glc-6-P, 0.2 unit of Glc-6-P dehydrogenase, and 0 (), 3 (), 5 (), 10 (), and 20 µM () PA. The residual C4H activity was then assayed as described in ``Materials and Methods''. Data correspond to means of duplicates. Inset, Times of one-half inactivation (t1/2) estimated from linear regression analysis were plotted against the reciprocal of PA concentration.

Other Methylenedioxy Compounds

View this table: [in this window] [in a new window]   Table I. Inactivation of CYP73A1 by methylenedioxy compounds CYP73A1 in transformed yeast microsomes were preincubated at a concentration of 250 nM with 4 mM Glc-6-P, 0.1 unit of Glc-6-P dehydrogenase, 100 µM NADPH, and 100 µM of each inhibitor. After 15 min, 10 µL of the mixture was transferred into the C4H assay for determination of residual activity. The assay contained 4 mM Glc-6-P, 0.1 unit of Glc-6-P dehydrogenase, 100 µM NADPH, and 100 µM radiolabeled trans-cinnamic acid in a final volume of 200 µL. 4-Coumaric acid formed after 3 min of incubation was determined as described previously (Reichhart et al., 1980).

Selectivity of PA toward C4H

A similar approach was proposed by Franklin (1991) to determine the proportion of P450 isozymes capable of oxidatively metabolizing macrolide antibiotics to a nitroso intermediate in liver microsomes. To determine an extinction coefficient () for the Soret maximum of the reduced CYP73A1-MI complex, we measured A₄₂₇₄₉₀ for several dilutions of yeast microsomes (i.e. 30-150 nM CYP73A1) after 15 min of incubation with 50 µM PA and a NADPH-regenerating system and subsequent complete reduction with sodium dithionite. A₄₂₇₄₉₀ was proportional to the CYP73A1 content of the incubation medium: the ₄₂₇₄₉₀ was 102 mM¹ cm¹. This extinction coefficient was then used to determine the P450-MI content (Table II) in microsomes of Jerusalem artichoke tubers treated to modify P450 enzyme subpopulations (Batard et al., 1995, 1997; Cabello-Hurtado et al., 1998b). CYP73A1 contents were determined in the same microsomes from the substrate-binding spectra (Urban et al., 1994). The difference spectra observed upon incubation of plant microsomes with PA and NADPH were identical to those obtained with recombinant CYP73A1. Table II shows that the C4H contents deduced from substrate binding were very similar to the content of P450-MI complexes measured in the same microsomes. Only the results obtained with microsomes prepared from aminopyrine-treated tissues suggested the possible existence of another P450 subspecies able to form a MI complex with PA.

View this table: [in this window] [in a new window]   Table II. Determination of the amounts of CYP73A1-MI complex formed in Jerusalem artichoke tuber microsomes after incubation with PA Microsomes were prepared from tuber tissues, sliced, and aged for 24 h in water (wounding) or 1 mM CuCl2, for 48 h in 20 mM aminopyrine, or for 72 h in 25 mM MnCl2, pH 7.0. All measurements were performed by differential spectrophotometry. Microsomes were diluted in 0.1 M sodium phosphate buffer, pH 7.4. Total P450 content was estimated from the reduced-CO binding spectra according to the method of Omura and Sato (1964). The C4H content was estimated from trans-cinnamic acid binding to native microsomes as previously described (Urban et al, 1994). The MI complex was quantified as described in ``Results''. All values are means of triplicates.

To further confirm the selectivity of PA, we tested if other available yeast-expressed plant P450 enzymes, CYP81B1 and CYP76B1 from Jerusalem artichoke, CYP51 from wheat, and CYP86A1 from Arabidopsis, were able to form MI complexes with PA. No MI-complex formation was detected when yeast microsomes containing from 68 to 125 pmol mg¹ protein of either P450 were incubated for 15 min with concentrations up to 100 µM PA (data not shown).

Effect of PA on the Phenylpropanoid Metabolism in Vivo

A BY2 tobacco cell suspension was simultaneously elicited with -megaspermin and given 100 µM PA. The cinnamic and 4-coumaric acid contents of the cells were determined 13 h after the beginning of the treatment (Fig. 7). The sharp decrease in 4-coumaric acid (13-fold), together with the strong cinnamic acid accumulation (20-fold) observed relative to control cells treated with elicitor alone, were consistent with an effective inhibition of C4H.

View larger version (28K): [in this window] [in a new window]   Figure 7. Effect of PA treatment on cinnamic and 4-coumaric acids concentrations in elicited tobacco cells. A synchronized BY2 tobacco cell suspension was treated with 50 nm -megaspermin, alone or together with 100 µM PA. Total cell contents in cinnamic and 4-coumaric acids (free plus conjugated forms) were determined after 13 h of treatment, as described in ``Materials and Methods''.

Scopoletin is a simple 7-hydroxylated coumarin. Its biosynthetic pathway is not elucidated, but involves at least the first two steps of the core phenylpropanoid pathway (Fritig et al., 1970). Scopoletin accumulation is observed in cultured tobacco cells in response to fungal elicitor. In tobacco leaves scopoletin is responsible for the fluorescent rings observed around local lesions occurring in the hypersensitive response to TMV. When plant cells were treated simultaneously with -megaspermin and PA, scopoletin accumulation was considerably decreased compared with cells treated with -megaspermin alone (Table III). A similar effect was observed when 500 µM PA was injected in TMV-infected tobacco leaves. Scopoletin accumulation measured after 3 d was decreased by about 70% compared with leaves injected with water (Table III).

View this table: [in this window] [in a new window]   Table III. Effect of PA on the induced accumulation of scopoletin in plant cells or tissues Synchronized BY2 tobacco cell suspensions were treated with 50 nM -megaspermin, alone (PA) or together with 100 µM PA (+PA). The scopoletin content of the cells was analyzed 13 h after treatment. Tobacco leaves were syringe infiltrated with water or 500 µM PA 16 h after inoculation with TMV. Scopoletin content of the tissues was determined 3 d after inoculation. Results are given in relative units compared with controls without elicitor treatment or TMV inoculation. Scopoletin contents in control cells and tissues were 0.11 and 0.12 µg g1 fresh weight, respectively.

	DISCUSSION

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Based on a structure-activity study we have recently characterized strong competitive inhibitors of C4H (Schalk et al., 1997). Mechanism-based inactivators are potentially more efficient and selective, since (a) they must satisfy the structural constraints imposed for binding to the active site, (b) the inhibitor must be acceptable as a substrate for its catalytical activation, and (c) reactive species produced by catalysis irreversibly or quasi-irreversibly alter the enzyme and remove it permanently or quasi-permanently from the catalytic pool (Ortiz de Montellano and Correia, 1995).

First used empirically as herbicide synergists or drug potentiators, MDP compounds have subsequently been shown to exert their activity via mechanism-based inactivation of xenobiotic-metabolizing P450 enzymes. The inhibition of P450 activities by MDP compounds and the mechanism-based formation of MI complexes have been extensively studied in a number of species both in vitro and in vivo and found to affect a broad range of P450-dependent reactions (Franklin, 1971; Hodgson and Philpot, 1974; Elcombe et al., 1975). Inhibition of P450 by MDP compounds has also been observed in plant tissues (Varsano et al., 1992; Kusukawa et al., 1995). It is generally accepted that the P450 inactivation and the unusual optical absorption spectra observed with inactivated enzymes result from the direct interaction with heme iron of a carbene formed by hydrogen abstraction or by elimination of water from a hydroxymethylene intermediate.

The strong -acceptor character of the carbene explains the shift of the Soret peak toward long-absorption wavelengths and the stability of the complex that is not displaced by CO or dioxygen (Wilkinson et al., 1984). MDP-P450 complexes are preserved through microsome extraction, dialysis, detergent treatment (Elcombe et al., 1975), oxidation, and re-reduction of the enzyme (Franklin, 1979). Inactivation, however, is not completely irreversible since the carbene can be displaced by type-I ligands (i.e. substrates), in particular once the enzyme is re-oxidized. There is a considerable degree of selectivity in the inhibitory potency of the MDP compounds toward different P450 enzymes (Wilkinson et al., 1984; Murray et al., 1993). The interactions of MDP side chains and aromatic cycles with the protein regions adjacent to the heme determine not only the binding of the compound prior to metabolism, but also the stability and further transformation of the complex.

In vitro, PA shows all of the characteristics of a specific ligand and of a very potent mechanism-based inactivator of C4H. The totality of the recombinant enzyme is complexed after less than 10 min in the presence of 30 µM PA, with a quasi-total loss in catalytic activity. Inactivation and formation of the MI complex are observed only in the presence of NADPH, increase in a time-dependent manner, and exhibit saturation kinetics. The presence of substrate or competitive inhibitors slows down or completely reverses the formation of the complex. This reversion process is, however, slow compared with inactivation and requires high, most likely nonphysiological, substrate concentrations.

The optical difference spectrum resulting from incubation of CYP73A1 with PA in the presence of NADPH is typical of a P450-MDP metabolite interaction (Wilkinson et al., 1984). This spectrum differs slightly from the typical type-III spectrum observed with the majority of MDP derivatives, since the peak at 455 nm appears only very transiently. The two peaks detected in the MI-bound versus free-P450 difference spectra are thought to reflect the formation of two distinct types of MI-P450 complexes, but the exact nature of the two complexes has not been elucidated. The 455-nm peak is usually considered to reflect only the interaction of the carbene with heme iron. Conversion of the 455-nm-absorbing entity into a 427-nm-absorbing complex seems to depend both on the substituents of the MDP molecules and on the P450 isoform that metabolizes it.

It has been proposed that the 427-nm peak could result from interactions of the MDP side chains with the protein in the vicinity of heme or from a shift in the spin state of the heme iron. The most plausible explanation, however, seems to be a rupture of the bond between heme iron and its Cys thiolate fifth ligand (Dahl and Hodgson, 1979; Wilkinson et al., 1984). It can be speculated that this rupture results from a stretching of the iron-sulfur bond induced by the electron-withdrawing properties of MDP substituants or by the interactions of the MDP side chains with the protein. An increase in the iron-sulfur distance upon CO binding has been demonstrated for P450_cam (Raag and Poulos, 1989). In addition, the strong electrostatic interactions that are likely to ensure PA binding to the apoprotein and the shortened ring structure of the molecule compared with an ideal substrate (Fig. 2) would support the hypothesis of a very stretched complex. It has been reported that the presence of electron-withdrawing substituants on the MDP aromatic ring tends to favor the production of CO from the methylenic carbon of the methylenedioxy ring (Yu et al., 1980), leading to the formation of a typical complex absorbing at 450 nm. It is interesting to note that despite the presence of a carboxylic function on PA, no formation of a CYP73A1-CO complex was detected.

Its close structural analogy to the natural substrate seems to confer on PA a real selectivity for C4H. None of the other plant P450s that we tested so far, which were reported as fatty acid hydroxylases or sterol or 7-ethoxycoumarin O-dealkylases, were capable of forming spectrally detectable complexes that would indicate enzyme inactivation. PA was also apparently selective for C4H in microsomes from Jerusalem artichoke. However, the potential number of P450s in plant tissues is extremely high. In the phenylpropanoid pathway alone, more than 16 P450-dependent reactions have been characterized, and more than 120 plant P450 genes have already been isolated. C4H is very abundant in plants, as shown in Jerusalem artichoke tissues (Table II). Formation of a MI complex with a minor P450 subpopulation in plant microsomes would not be detectable by the spectrophotometric approach used in this work. It is thus reasonable to assume that PA is not strictly specific to C4H, although inactivation of another P450 isoform with a similar efficiency is highly improbable.

PA also seems to be an efficient inactivator of C4H in vivo, blocking the synthesis of 4-coumaric acid and inducing the accumulation of cinnamic acid in plant cells. Tools were already available to block the steps upstream and downstream in the pathway (Fig. 1). -Aminooxy--phenylpropionic acid, which inhibits Phe-ammonia lyase, has been available and used extensively for many years to block the whole phenylpropanoid pathway (Amrhein and Gödeke, 1977). 3,4-(Methylenedioxy)-cinnamic acid has recently been described as a potent inhibitor of the coumaroyl CoA-ligase (Funk and Brodelius, 1990). This compound, which also bears a methylenedioxy function and is a structural analog of cinnamic acid, does not affect C4H. We have previously shown that size and charge are critical determinants in the binding of ligands into the substrate pocket of CYP73A1 (Schalk et al., 1997). This is confirmed by the data presented in Table I. It is thus probable that the methylenedioxy ring renders the molecule too large to bind or to have access to the active site of C4H. MDCA remains a potential inhibitor of other P450 enzymes downstream in the pathway.

Compared with aminooxy--phenylpropionic acid and 3,4-(methylenedioxy)-cinnamic acid, PA offers new potential applications. It can be used to selectively block the C6-C3 metabolism and, possibly, to shift the precursor flow into the formation of C6-C1 compounds such as benzoic and salicylic acids. It also offers a possibility to determine if 4-hydroxybenzoic acids derive from cinnamic acid or must be formed from 4-hydroxycinnamic acids. Many reports (Werck-Reichhart, 1995; Sewalt et al., 1997) have suggested that C4H, possibly via its product and substrate, has a regulatory function in the coordinate regulation of the phenypropanoid pathway. PA is a new tool to test this hypothesis.

	FOOTNOTES

   Received February 17, 1998; accepted May 21, 1998.

	ABBREVIATIONS

Abbreviations: C4H, trans-cinnamate 4-hydroxylase (NADPH:oxygen oxidoreductase [4-hydroxylating] EC 1.14.13.11) . MDP, methylenedioxyphenyl. MI, metabolic intermediate. PA, piperonylic acid. TMV, tobacco mosaic virus.

	ACKNOWLEDGMENTS

We thank Drs. D. Pompon and P. Urban for providing the W(R) yeast strain and the pYeV60 expression vector and Dr. I. Benveniste for microsomes of yeast expressing CYP86A1. We also thank M.F. Castaldi for her technical assistance. The critical reading of the manuscript by F. Bernier and R. Kahn is gratefully acknowledged.

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