ORIGINAL ARTICLE

Irene Benveniste Æ Roberte Bronner Æ Yong Wang

Vincent Compagnon Æ Pierre Michler

Lukas Schreiber Æ Jean-Pierre Salaun

Francis Durst Æ Franck Pinot

CYP94A1, a plant cytochrome P450-catalyzing fatty acidx-hydroxylase, is selectively induced by chemical stressin Vicia sativa seedlings

Received: 10 October 2004 / Accepted: 21 January 2005 / Published online: 21 May 2005� Springer-Verlag 2005

Abstract CYP94A1 is a cytochrome P450 (P450) cata-lyzing fatty acid (FA) x-hydroxylation in Vicia sativaseedlings. To study the physiological role of this FAmonooxygenase, we report here on its regulation at thetranscriptional level (Northern blot). Transcripts ofCYP94A1, as those of two other P450-dependent FAhydroxylases (CYP94A2 and CYP94A3) from V. sativa,are barely detectable during the early development of theseedlings. CYP94A1 transcripts, in contrast to those ofthe two other isoforms, are rapidly (less than 20 min)and strongly (more than 100 times) enhanced aftertreatment by clofibrate, an hypolipidemic drug in ani-mals and an antiauxin (p-chlorophenoxyisobutyric acid)in plants, by auxins (2,4-dichlorophenoxyacetic acid andindole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid.All these compounds activate CYP94A1 transcriptiononly at high concentrations (50–500 lM range). In

parallel, these high levels of clofibrate and auxins modifyseedling growth and development. Therefore, theexpression of CYP94A1 under these conditions and theconcomitant morphological and cytological modifica-tions would suggest the implication of this P450 in aprocess of plant defense against chemical injury.

Keywords Chemical transcription activation ÆDefense Æ Fatty acid hydroxylases Æ Stress Æ Vicia

Abbreviations: 2,3-D: 2,3-Dichlorophenoxyaceticacid Æ 2,4-D: 2,4-Dichlorophenoxyacetic acid Æ FA:Fatty acid Æ GST: Glutathione-S-transferase Æ IAA:Indole-3-acetic acid Æ P450: Cytochrome P450 Æ PCIB:p-Chlorophenoxyisobutyric acid Æ PMSF:Phenylmethane sulfonylfluoride

Introduction

Cytochromes P450 hydroxylating fatty acids (FAs) areencoded by multiple genes in fungi, animals and plants.In yeast strains assimilating hydrocarbons (e.g., Candidamaltosa), several cytochromes P450 (P450s), belongingto the CYP52 phylogenetic family, oxidize differentchain length n-alkanes and FAs at their terminal methylgroup, leading respectively to the corresponding FAsand diacids after two additional sequential oxidationsteps catalyzed by an alcohol- and an aldehyde-dehy-drogenase (Scheller et al. 1996). The FA and the diacidso formed are then subjected to peroxisomal b-oxidationto produce energy for cell metabolism.

In animals, saturated FAs with chain lengths rangingfrom C12 to C20 and unsaturated FAs are hydroxylatedpredominantly at the terminal carbon. These hydroxy-lations are mainly catalyzed by P450s from the CYP4family (Simpson 1997; Okita and Okita 2001). The earlyrole of the terminal oxidation of FAs assigned to these

I. Benveniste Æ R. Bronner Æ Y. Wang Æ V. CompagnonP. Michler Æ L. Schreiber Æ J.-P. Salaun Æ F. Durst Æ F. PinotCNRS - Institut de Biologie Moleculaire des Plantes - UPR2357 -Departement �Reponses metaboliques a l’environnementbiotique�, Universite Louis-Pasteur,28 Rue Goethe, 67083 Strasbourg Cedex, France

I. Benveniste (&)IBMP-Institut de Botanique, 28 Rue Goethe,67083 Strasbourg Cedex, FranceE-mail: Irene.Benveniste@bota-ulp.u-strasbg.frTel.: +33-3-90241878Fax: +33-3-90241921

Present address: Y. WangInstitut d’Ecologie-Biologie et Physiologie Vegetales,Universite de Lausanne, CH, 1015 Lausanne, Switzerland

Present address: L. SchreiberBotanisches Institut und Botanischer Garten, Kirschallee 1,53115 Bonn, Germany

Present address: J.-P. SalaunBiochimie et Biologie Moleculaire-Faculte de Medecine-CS,93837-29238 Brest Cedex3, France

Planta (2005) 221: 881–890DOI 10.1007/s00425-005-1503-y

enzymes was the maintenance of FAs homeostasis(Gibson 1989). Clofibrate, an hypolipidemic drug forhumans, activates transcription of genes involved in lipidhomeostasis, such as CYP4 isoforms, acyl CoA oxidaseand the peroxisomal b-oxidation system. Later on, theimportance of oxidized FAs, especially of arachidonicacid, was described in the regulation of crucial functionsin animals (e.g., inflammation, kidney ion exchanges)(Helvig et al. 1998; Capdevila et al. 2002). In addition, arole of signal production was ascribed to some of theisoforms of the CYP4 family (Cowart et al. 2002).

Recently, multiple isoforms of P450-dependent FAhydroxylases have been identified in plants and thesubstrate specificities of some have been investigated.Vicia sativa seedlings were shown to contain at leastthree isoforms (CYP94A1, 94A2 and 94A3), eachhydroxylating a selective panel of FAs and exhibitingindividual regulations (Tijet et al. 1998; Le Bouquinet al. 1999). Three genes from Nicotiana tabacumbelonging to the same CYP94A subfamily have beencloned (94A4, 94A5 and 94A6) and the correspondingenzymes expressed in yeast displayed specific patterns ofFAs oxidations (Le Bouquin et al. 2001). The first genecoding for an Arabidopsis thaliana FA x-hydroxylase(CYP86A1) was cloned out of an A. thaliana EST libraryusing consensus sequences derived from mammalianCYP4 and yeast CYP52 families (Benveniste et al. 1998).Subsequently, several P450 subfamilies, each includingmultiple isoforms, phylogenetically related to genescoding for known FA hydroxylases, were identified inthe complete A. thaliana genome sequence. Examinationof EST libraries and genomic sequences from soybean,Medicago truncatula, tomato, rice, wheat, etc, suggeststhe existence of multiple P450s capable of FA hydrox-ylation in a large variety of plant species.

These observations raise the questions of the biolog-ical roles of these distinct P450 isoforms and why plantsduring their evolution have developed so many FA hy-droxylases. The presence of multiple molecular speciessuggests diverse functions for these enzymes and severalpotential biological functions have been proposed. Re-lease of free FAs by activation of phospholipases hasbeen reported in the early responses of plants to differentstresses, such as exogenous high auxin concentrationsapplication (Paul et al. 1998) and TMV infection of aresistant tobacco (Dhondt et al. 2000). Therefore, x-hydroxylation of FAs could be a first step in the detox-ification of deleterious free FAs. Further sequential oxi-dations of the terminal hydroxyl group of the FAs, byalcohol- and aldehyde-dehydrogenases and by dedicatedP450 isoforms (Le Bouquin et al. 2001), result in thecorresponding diacids, which are then efficiently chain-shortened by peroxisomal and mitochondrial b-oxida-tion pathways leading to energy production and themaintenance of the cellular homeostasis of free FAs.

On the other hand, the capability of FA hydroxy-lases (Benveniste et al. 1998; Tijet et al. 1998; Pinotet al. 1999, 2000) to synthesize most of the monomersfound in cutin and suberin suggests their involvement

in the plant defense mechanisms, by reinforcing orfixing (Kolattukudy 1980; Holloway 1982) these pro-tective polymers. Interestingly, in agreement with thisrole, a mutation by transposon insertion into theCYP86A8 gene, coding for a P450 x-hydroxylatingFAs ranging from C12 to C18:1 in A. thaliana, re-sulted in the abnormal fusion of rosette leaves due tothe absence of cutin on the outside of epidermal cellsthat induces sutures between cell walls of contiguousorgans (Wellesen et al. 2001). Complementation of theinsertional mutant with the CYP86A8 gene restoredthe wild phenotype. Noteworthy, the presence in theseplants of CYP86A1, which shows a similar substratespecificity (Benveniste et al. 1998) as CYP86A8, didnot rescue the mutation of CYP86A8, indicating thatclosely related members of this P450 subfamily are notredundant. This is likely due to a different organ- ortissue-localization, or to a different temporal expres-sion of these isoforms.

Another physiological function of FA hydroxylasescould be to produce secondary messengers involved inthe resistance of plants against pathogens. Recent resultsshow the protective effects of several hydroxylated FAsagainst fungal infection: the presence of an hydroxylgroup at the omega end appears to reinforce the effi-ciency of oxygenated FAs in the protection of the plant(Schweizer et al. 1996a, b; Wang et al. 2000).

Selective induction, often by the activation of genetranscription, constitutes a remarkable property of thedifferent isoforms of P450. Thus, in animals, variousCYP4 isoforms which hydroxylate FAs and prosta-glandins (Simpson 1997) are selectively induced byendogenous (FAs, prostaglandins) and exogenous (per-oxisome proliferators such as clofibrate) hydrophobiccompounds, and their mechanisms of regulation are nowlargely deciphered (Berger and Moller 2002).

In plants, our laboratory has extensively studied theexpression of FA hydroxylases from V. sativa seedlings(Tijet et al. 1998; Pinot et al. 1998; Le Bouquin et al.1999). During germination and the early stages ofdevelopment, the seedlings expressed low constitutivelevels of mRNAs and displayed low FAs hydroxylaseactivities. However, CYP94A1 exhibited a selective andsignificant increase of its gene expression and activity inresponse to exogenously applied compounds, such asclofibrate (Tijet et al. 1998) and methyljasmonate (Pinotet al. 1998). A better knowledge of the potential inducersand the finemechanisms of its regulation should elucidatethe physiological functions of CYP94A1 in V. sativa.

Here, we describe the dose effect of different in-ducers (clofibrate, auxins, inactive auxin analog, sali-cylic acid (SA)) on the mRNA level of CYP94A1 and/or the enzyme activity. We show that only relativelyhigh concentrations of inducers, which most oftenmodify the growth of the seedlings, rapidly anddrastically enhance the expression of CYP94A1. Theseresults suggest the involvement of this P450 in pro-cesses involved in responses of V. sativa seedlings tostress.

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Materials and methods

Plant material and treatments

Vetsch seeds (V. sativa, var Lolita) were obtained fromBlondeau (Bersee, France). Seeds were germinated andgrown on moist filter paper at 25�C in the dark. Seedcoats were removed from 3-day-old seedlings which werethen aerated with compressed air in aqueous solutions ofdifferent compounds. The clofibrate emulsion in waterwas maintained by bubbling. Salicylic acid was dissolvedin water as potassium salt. Seedlings were harvestedafter different times of treatments, frozen in liquidnitrogen, and stored at �30�C until microsome prepa-ration or RNA extraction.

In order to quantify the CYP94A1 mRNA in organsnot directly in contact with the inducer, etiolated seed-lings were germinated in glass dishes for 3 days, thentreated by clofibrate only at the level of the roots. Thedifferent parts of the seedlings were harvested separately(roots, first and second epicotyl internodes, apical bud)after 20 h of treatment.

Northern blot analysis

Total RNAs from whole seedlings (5 g), includingcotyledons removed from their seed coat, were ex-tracted after the modified hot borate method describedby Wan and Wilkins (1994). Total RNAs from dif-ferent organs (2 g) of the seedlings were prepared asdescribed in Goodall et al. (1990). For Northern blotanalysis, total RNAs (20 lg/lane) were denatured,subjected to electrophoretic separation on 1.2% aga-rose gel containing formaldehyde and transferred ontoa Hybond N+ membrane (Amersham). RNAs werelinked to the membrane by baking for 2 h at 80�C.The blot was hybridized with the 32P-labeledCYP94A1 coding region, at 65�C for 16 h. A radish18S ribosomal cDNA was used as an internal stan-dard. After hybridization, the blot was washed twicewith 2X SSC (1X SSC: 0.15 M NaCl/0.015 M sodiumcitrate)/0.1% SDS at room temperature for 15 min,and twice with 0.2X SSC/0.1% SDS at 55�C for30 min. Densitometric quantification of mRNA wasperformed from scanned autoradiography (Agfa ArcusII Scanner) using the free NIH-Image program version1.5.9. Spot-intensity measurement of mRNA was ad-justed as a function of the intensity of the internalstandard. 32P-radioactivity was also quantified with aFujix Bas 1000 Phosphorimager using the MacBASV2.2 program.

Preparation of V. sativa microsomes

Etiolated seedlings (5 g) treated by different chemicals fordifferent times were homogenized with an Ultra-Turrax

homogenizer (15,000 rev./min, twice for 30 s each) onice, in a final volume of 50 ml of 100 mM sodium phos-phate buffer, pH 7.4, containing 250 mM sucrose,40 mM sodium ascorbate, 10 mM 2-mercaptoethanoland 1 mM phenylmethane sulfonylfluoride (PMSF).The homogenate was filtered through 50 lmBlutex clothand centrifuged for 10 min at 10,000 g. The resultingsupernatant was centrifuged for 1 h at 100,000 g yieldingthe microsomal pellet. Microsomes were resuspendedin 2 ml of 20 mM sodium phosphate buffer, pH 7.4,containing 30% (v/v) glycerol, homogenized with aPotter-Elvehjem homogenizer, divided into aliquotsand stored at �30�C. Protein concentrations were esti-mated with a microassay from Bio-Rad, with BSA as astandard.

Lauric acid x-hydroxylase activity

Radiolabeled (1�14C)-lauric acid from Amersham iso-topically diluted with cold lauric acid from Sigma weredissolved in ethanol, which was evaporated off beforethe addition of microsomes (50 ll) to the glass tube.Resolubilization of the substrate was confirmed bymeasuring the radioactivity of the incubation medium.

CYP94A1 laurate x-hydroxylase activity wasdetermined by following the rate of formation of thehydroxylated product. The standard assay (0.2 ml)contained 20 mM sodium phosphate buffer, pH 7.4,containing 1 mM NADPH and radiolabeled laurate(55 lM). The reactions were initiated by the additionof NADPH, allowed to proceed for 15 min at 27�C,and stopped by the addition of 0.1 ml of acetonitrile(containing 0.2% acetic acid). The reaction productswere resolved by TLC: incubation media were directlyspotted on TLC plates, which were developed with amixture of diethyl ether/light petroleum (boiling range40–60�C)/formic acid (50:50:1, v/v/v). The plates werescanned by a thin-layer scanner (Berthold LB 2723),and the area corresponding to the metabolite wasscraped into a counting vial and quantified by liquidscintillation.

Cytological observations

Thin transversal hand sections of the top of the epicotylfrom etiolated V. sativa seedlings were observed byepifluorescence after auramine o staining (0.01% aur-amine o solution in Tris/HCl 0.05 M, pH 7.2, for 5 min)(Heslop-Harrison 1977). This staining reveals lipidicstructures and therefore waxes and cutin.

Transversal hand sections of the root were treated byphloroglucinol (hydrochloric solution) to stain ligninand lignin-like structures, or by the red lipophilic SudanIII dye (0.3% solution in 70% ethanol) to observe lipidiccompounds such as suberin.

Strips of epidermal cells were observed withoutstaining by photonic microscopy.

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Transmission electron microscopy (TEM)

Epicotyl tissues were fixed overnight by 5% glutaralde-hyde in 0.1 M Na-phosphate buffer, pH 7.2, at 0�C,followed by an additional fixation by 1% osmiumtetroxide in solution in the former buffer, at 0�C, for 2 h.Dehydration process of the fixed biological materialoccured at room temperature, before preinclusion intoEpon resin, in the presence of propylene oxide. Inclusionin Epon occured overnight at room temperature andpolymerization at 60�C for 60 h. TEM observationswere carried out on transversal sections of 90 nm.

Results

Effects of clofibrate on V. sativa seedlings

Clofibrate (2-(p-chlorophenoxy)-2-methyl-propionic acidethyl ester), a potent inducer of FAs x-hydroxylases inanimals, is also highly efficient in the activation oftranscription of CYP94A1 coding a FAs x-hydroxylasefrom V. sativa seedlings. The mechanisms by whichclofibrate is perceived by V. sativa seedlings and thesignal transduction pathway up to gene activation re-main unclear.

Effect on the expression of CYP94A1

Figure 1 shows the evolution of the transcripts ofCYP94A1 from V. sativa seedlings after treatment bydifferent concentrations of clofibrate. Three-day-old

etiolated seedlings were aerated for different times inclofibrate emulsions or in water. Transcripts ofCYP94A1 from control seedlings were nearly undetect-able on Northern blot. On account of the low level ofthese transcripts, the control data are not representedhere, since their autoradiographic visualization needslonger exposition times than transcripts from treatedseedlings, hindering therefore accurate comparativequantifications. In contrast, incubation of the seedlingsin the presence of increasing concentrations (50, 100, 200and 500 lM) of clofibrate produced a rapid increase ofthe steady state level of CYP94A1 transcripts, reaching amaximum after 6 h. The level of induction was depen-dent on the concentration of the inducer. The lowestconcentrations (50 lM and 100 lM) increased tran-siently the level of CYP94A1 mRNAs over 24 h. Higherclofibrate concentrations (200 lM and 500 lM) led to asecond wave of induction of these mRNAs.

Effect on morphology and cytologyof V. sativa seedlings

The morphology of clofibrate-treated seedlings wasclearly different from that of the control. Figure 2 pre-sents the inhibitory effect of increasing concentrations ofclofibrate on the length of different organs (primaryroot, epicotyl internodes, apical bud). The most visibleeffect was a shortening of the epicotyl, especially of thesecond and third internodes which are most rapidlyelongating at this stage of normal development. Inaddition, clofibrate also inhibited the development ofsecondary roots and, as a later effect, resulted in the loss

Fig. 1 Evolution of CYP94A1 transcripts in V. sativa seedlingstreated by different concentrations of clofibrate for different times.Estimation by autoradiogram scanning of CYP94A1 transcripts onNorthern blots of 20 lg total RNAs, electrophoretically separatedon a denaturating formaldehyde 1.2% agarose gel and transferredby capillarity onto Hybond N+ membrane, from 3-day-oldetiolated seedlings treated for different times with differentconcentrations of clofibrate (filled triangle) 0.05 mM; (open circle)0.1 mM; (open square) 0.2 mM; (filled square) 0.5 mM. Thedensitometric values of the radish 18S rRNA were used for thecalibration. Results are means of duplicate measurements

Fig. 2 Inhibition of the elongation of different organs of V. sativaseedlings by increasing concentrations of clofibrate. Three-day-oldetiolated seedlings were treated by bubbling in different concen-trations (0.05, 0.1, 0.2 and 0.5 mM) of clofibrate solutions or inwater for the control, for 52 h. Inhibition by clofibrate wascalculated by comparison of the different organs length with that ofwater control: (open circle) root, (closed circle) first more basalepicotyl internode, (open square) second internode, (filled square)third internode, (open triangle) apical bud. Each value representsthe average of 30 independent measurements

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of apical dominance with the growth of axillary meris-tems.

Cytological studies on cross sections of the stem werecarried out at the level of the second internode, whereclofibrate effect was most marked. At this place, thick-ening of the epidermal cells was observed, comparedwith the control (Fig. 3a, b). In addition, increasedthickness of the cuticle of epidermal cell was clearlyvisible. In parallel, this thickening was accompanied bythe inhibition of cell elongation, easily observed on thestrips of stem epidermal cells (Fig. 3c, d). On the otherhand, the size of the epidermal cells from primordialleaves was not altered by the treatment (Fig. 3e, f).Clofibrate also modified the stem vascular bundle,retarding the development of the xylem vessel. Anothernoteworthy effect of clofibrate was observed on the rootendodermis. Endodermal cells are considered to formapoplastic transport barriers in roots toward water,solutes or pathogens (Schreiber et al. 1999). In com-parison to control seedlings, treated plants had enlargedendodermal cells, exhibiting well developed Casparianbands, enriched in lignin, as vizualized after phloroglu-cinol staining (Fig. 3g, h), and surrounded by a suberin-enriched layer, revealed by Sudan III staining (Fig. 3i, j).This enlargement of the endodermal cells by clofibrateshould be an event of the defense reaction of the seedlingagainst the applied exogenous compound. x-HydroxyFAs represent an important part of the aliphaticmonomers of suberin (Zeier et al. 1999). Therefore,based on the previous cytological observations and thecatalytic capacities of CYP94A1, this P450 could belocally expressed in the endodermis and involved in thebiosynthesis of suberin. This assumption should beelucidated by future in situ hybridization studies.

Electron microscopy observations of transversal sec-tions of the top of epicotyl from both control (Fig. 4a)and treated plants (Fig. 4b) showed a distinct increase inthe thickness of the wall and cuticle of the epidermalcells resulting from clofibrate application. However,quantification by GC/MS of cutin monomers revealedthat the cuticle of control and treated seedlings con-tained an equal amount of monomers per surface unit.Since clofibrate application resulted in growth reductionof the epicotyl (as shown above), an equal amount ofcutin was deposited over a smaller surface in a thickerlayer. Therefore, CYP94A1 would not constitute a lim-iting step in cutin biosynthesis, in spite of its highcapability to produce cutin monomers.

Organ selective induction of CYP94A1after clofibrate treatment

To evaluate the biological function of this inducible FAhydroxylase, the localization of its transcripts in differentorgans of clofibrate-treated V. sativa seedlings wasinvestigated by Northern blot analysis. After 3 days ofgermination on water, only the roots of the seedlingswere soaked in an aqueous solution of 500 lM clofibrate

for 46 h. Then, roots, first and second stem internodesand apical buds were harvested separately and the totalRNAs were extracted from these organs. The Northern

Fig. 3 Effect of clofibrate (0.5 mM) on the cytology of V. sativaseedlings. Epifluorescence observation of the epicotyl epidermalcells of control (a) and 18 h clofibrate-treated seedlings (b) onauramine-stained transversal hand sections of the top of theepicotyl: thickness of the epidermal cells is increased, in addition tothickening of the cell wall and the cuticle (ep epidermis; c corticalcells). Comparison of the length of epicotyl epidermal cells,observed on epidermal strips from the basis of the second internodefrom water control (c) and 18 h 0.5 mM clofibrate-treated seedlings(d). Comparison of the length of primordial leaf epidermal cellsfrom control (e) and clofibrate-treated plants (f), at the samedevelopment stage. g–j Views focus on the root endodermis,indicating the clofibrate-enlarged endodermis cells, after phloro-glucinol (h) and Sudan III (j) stainings, compared with the controls,respectively (g) and (i); (en endodermis; c cortical cells; cc centralcylinder). Scale bar 50 lm

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blot represented in Fig. 5 compares the levels ofCYP94A1 transcripts in control and treated seedlings:CYP94A1 mRNAs were barely detectable in controltissues, but were increased by clofibrate treatment in allthe organs checked. Noteworthy, induction was highestin the second internode located just under the apical bud,precisely where the growth inhibition was strongest.

Effects of 2,4-dichlorophenoxyacetic acid (2,4-D)on V. sativa seedlings

Several of the above morphological and cytologicalobservations (inhibition of secondary root development,loss of apical dominance, inhibition of cell elongation,delay of xylem development) indicate that clofibrate actsas an auxin antagonist on V. sativa seedlings. Since the

chemical structure of clofibrate is close to that of thesynthetic auxin 2,4-D, we analyzed its effect on the levelof transcription of CYP94A1 and on the lauric acid x-hydroxylase activity of the corresponding enzyme.

Several concentrations of 2,4-D, ranging from hor-monal (1 lM) up to herbicidal (500 lM) doses, wereapplied on 3-day-old etiolated V. sativa seedlings, agi-tated by air bubbling in aqueous solutions of 2,4-D.Figure 6a presents the evolution of lauric acid hydrox-ylase activity, catalyzed by CYP94A1, after the treat-ment of the seedlings by different concentrations of2,4-D. A high concentration (500 lM) was required torapidly and strongly increase the enzyme activity, whichreached a maximum after approximately 24 h and thenslowly declined. At 100 lM, 2,4-D stimulated theactivity to a lesser extent. Lower concentrations of 2,4-D(10 and 1 lM) were ineffective to stimulate lauratehydroxylation activity.

These results were corroborated by the lack ofCYP94A1 transcript induction at low concentrations of2,4-D (1 lM and 10 lM), whereas, at 100 lM and par-ticularly at 500 lM 2,4-D, there was a rapid and impor-tant increase of the CYP94A1 transcript level (Fig. 6b).The steady state level of CYP94A1 RNAs peaked after6 h of a 100 lM 2,4-D treatment and 12 h of a 500 lM2,4-D treatment, and then decreased until 48 h.

At 10, 100 and especially 500 lM concentrations of2,4-D the morphology of the seedlings was modified.Elongation of the primary root was inhibited and aswelling of the upper part of the epicotyl, just beneaththe apical bud, was observed. At the highest concen-tration, the epicotyl epidermal cells were clearly enlargedand covered by a thicker cell wall. The number of vas-cular bundles was decreased, clearly indicating that 2,4-D at a 500-lM concentration stressed the seedlings.

Effects of 2,3-dichlorophenoxyacetic acid (2,3-D)on V. sativa seedlings

The capacity of the inactive auxin 2,3-D, a structuralanalog of clofibric acid and 2,4-D, to induce the

Fig. 4 Ultrastructure of the outside of an epidermal cell from theepicotyl top of etiolated 3-day-old V. sativa seedlings grown for20 h in water or in a 0.5 mM clofibrate solution. Transmissionelectron microscopy micrographs of transversal sections of epicotylepidermal cells: in comparison to the water control (a), clofibrate(b) increases clearly the thickness of the cell wall (w) and the cuticledeposition (c). These micrographs are representative of the stemepidermal cells under the two conditions. Scale bars 0.5 lm

Fig. 5 Accumulation of CYP94A1 mRNA in various organs ofcontrol (C) and clofibrate-treated (T) V. sativa seedlings. Three-day-old etiolated seedlings, directly germinated on glass dishes inthe presence of water, were freshly watered or treated with 0.5 mMclofibrate at the root level for 46 h. Northern blot hybridizationanalysis was performed on 20 lg of total RNAs fractionated byformaldehyde denaturating agarose gel electrophoresis and trans-ferred to a nylon membrane (Hybond N+). RNAs used were fromroots, first developing stem internode (1st IN), second developingstem internode (2nd IN) and apical bud (apex). The membrane wassequentially hybridized with the CYP94A1 and 18S rDNA probes

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transcription of CYP94A1 was investigated. Etiolated V.sativa seedlings were treated for 48 h with 100 lM and500 lM solutions and total RNAs were extracted afterdifferent times of treatment. Figure 7a presents the evo-lution of CYP94A1 transcripts: at 100 lM, 2,3-D pro-duced a rapid but transient increase of the transcriptswhich returned to their initial level of expression after 24 hof treatment; a 500-lM concentration produced a higherand longer lasting increase of the steady state level ofCYP94A1 mRNAs. Beside this large increase inCYP94A1 expression, treatment with 2,3-D produced astrong inhibition of the elongation of the seedling epi-

cotyl, similar to that observed in plants exposed to clofi-brate.

Effects of indole-3-acetic acid (IAA)on V. sativa seedlings

Induction of CYP94A1 transcripts by 2,4-D, a syntheticauxin, and even more effectively, by clofibrate, a struc-tural analog, and by 2,3-D, an inactive synthetic auxin,prompted us to investigate the effect of IAA, a naturalauxin, with a different chemical structure. Three-day-oldetiolated V. sativa seedlings were treated with threeconcentrations (50, 100 and 200 lM) of IAA for 48 h in

Fig. 6 Evolution of lauric acid x-hydroxylase activity (a) and ofCYP94A1 transcripts (b) from etiolated V. sativa seedlings treatedby different concentrations of 2,4-D for different times. Micro-somal lauric acid x-hydroxylase activity from water controlseedlings (filled circle) or from seedlings treated with 0.1 mM(filled triangle) or 0.5 mM (filled square) 2,4-D was expressed inpmoles/min/mg microsomal protein. Microsomes from 5 g seed-lings were resuspended in 2 ml resuspension buffer and 50 lL wereincubated for 15 min at 27�C in the presence of 1 mM NADPHand 55 lM (1�14C)-radiolabeled lauric acid. Activities are meansof six measurements. CYP94A1 transcripts from water controlseedlings (open circle), or from seedlings treated with 0.1 mM (opentriangle) or 0.5 mM (open square) 2,4-D were determined onNorthern blot after hybridization of a 32P-labelled CYP94A1 probeon 20 lg of total RNAs separated electrophoretically on aformaldehyde denaturating 1.2% agarose gel. Intensities of thespecific spots were measured with a Fuji Phosphorimager andquantified with the MacBAS V2.2 program, and represented asrelative units. Values are means of three quantification measure-ments

Fig. 7 Evolution of CYP94A1 transcripts from 3-day-old etiolatedV. sativa seedlings treated for different times with differentconcentrations of 2,3-D and IAA. Twenty microgram total RNAswere electrophoretically separated on a formaldehyde denaturating1.2% agarose gel, transferred on Hybond N+ membrane andhybridized with a 32P-labeled CYP94A1 probe. Northern blotquantification of CYP94A1 transcripts from a seedlings treatedwith 0.1 mM (open circle) and 0.5 mM (filled circle) 2,3-D; the levelof radioactivity was determined with a Fuji Phosphorimager asmentioned above. The values are means of three measurements.Northern blot quantification of CYP94A1 transcripts fromb seedlings treated with 0.05 mM (open circle), 0.1 mM (opensquare), 0.2 mM (filled circle) IAA and water control (filled square);quantification was performed by scanning the autoradiographicspots (Agfa Arcus II Scanner) using the free NIH-Image programversion 1.5.9. The values are means of three measurements

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aerated water solutions. Figure 7b shows the rapid andtransient increase of CYP94A1 transcripts: in contrast towater controls, transcripts from treated plants increasedafter 3 h, reached a maximum after 12 h, and declinedrapidly until 24 h treatment. The amplitude of inductionwas dependent on the concentration of IAA.

In parallel, at all the concentrations used, IAA pro-duced morphological modifications, similar to those al-ready observed with 2,4-D, consisting of less elongationand distension of the top of the epicotyl, and of defini-tive inhibition of the development of the epicotyl hookand the apical bud. In addition, the elongation of theprimary root was inhibited, and the growth of thenumerous secondary roots was stopped. Histologicalstudies showed that the normally ordered epicotyl epi-dermal layer was perturbed, with inhibited cell elonga-tion and some epidermal cells forming papilla. Inaddition, the epicotyl cortical cells were larger. In theroot, IAA favored the development of the endodermislayer, whereas the maturation of xylem in both epicotyland root was retarded.

The rapid and enhanced transcription initiation ofCYP94A1 in response to exogenous administration ofhigh concentrations of auxin analogs producing pertur-bations in the seedling development suggests that thisFA hydroxylase plays an active role in response tochemical stresses. However, the morphological changesinduced by auxins and auxin antagonists may be due tothe activation of multiple genes, possibly involved insignal transduction pathways, phenylpropanoid path-way, glutathione-S-transferase (GST), etc.

Effect of SA on CYP94A1 transcripts

The strong increase of CYP94A1 transcription in re-sponse to methyljasmonate application (Pinot et al.1998) suggests the involvement of this gene in plantpathogen interactions. Therefore the effect of SA, aplant pathogen signal (Shah and Klessig 1999), whichmay mimic a biotic stress, was investigated on thetranscription level of CYP94A1, to check the involve-ment of this gene in response to a pathogen attack.

Three-day-old etiolated seedlings were treated foreither 8 or 24 h with 0.1 mM and 1 mM potassiumsalicylate solutions. At both times of treatment, only thehighest concentration used produced a significantinduction of CYP94A1 transcripts, which were enhancedsixfold to sevenfold, over the water control. At the 0.1-mM concentration, the transcripts were barely above thecontrol. The development of the seedlings was not visi-bly perturbed by treatment with SA.

Effect of clofibrate, 2,4-D, 2,3-D, IAA and salicylateon CYP94A2 and CYP94A3 expression

CYP94A1 hydroxylates FAs with different chain lengthsranging from 10 to 18 atoms of carbon. In contrast,

CYP94A2 and CYP94A3, the two additional FA x-hydroxylases characterized in V. sativa seedlings, oxidizemost efficiently myristate and caprate, respectively. Wecompared the expression of these latter genes with thatof CYP94A1 by Northern blot analysis. CYP94A2 andCYP94A3 genes were barely undetectable during theearly development of the seedlings, as CYP94A1.However, contrary to CYP94A1, the transcription ofCYP94A2 and CYP94A3 was not modulated by theabove checked inducers, pointing out the selectiveinvolvement of CYP94A1 in response to abiotic andbiotic stresses.

Discussion

These results demonstrate the selective and dose-dependent induction of one isoform of FA x-hydroxy-lase (CYP94A1) from V. sativa seedlings, by exogenouscompound (clofibrate, 2,4-D, 2,3-D, IAA and SA)applications. This cyt P450-dependent x-hydroxylaseoxidizes a large panel of FAs ranging from 10 to 18carbon atoms in length, including saturated medium-chain FAs (C10 to C16) and unsaturated long-chain FAs(C18: 1, C18: 2 and C18: 3), nevertheless, with differentcatalytic efficiencies (Tijet et al. 1998). 9,10-Epoxystea-rate constitutes presently by far the best substrate (Pinotet al. 1999, 2000).

The very fast transcription activation of CYP94A1implies an efficient recognition of the above chemicalsand a rapid signal transduction up to the gene activa-tion. The increase of CYP94A1 transcripts is concen-tration-dependent, with a biphasic time course ofexpression. The lowest effective concentrations fortranscription activation (50 lM clofibrate or IAA and100 lM 2,4-D or 2,3-D) produced a rapid but transient(12 h) increase of CYP94A1 transcripts, whereas higherconcentrations (200 lM IAA and 500 lM clofibrate,2,4-D or 2,3-D) extended the induced level of geneexpression for more than 24 h. These different patternsof expression (early and late induction) suggest thatdifferent mechanisms might be involved in inducer per-ception, signal transduction or in the transcriptionactivation itself.

The rapid onset of transcription in response to thesechemicals is in favor of activation mechanisms (Harperet al. 1993 ; Van der Hoeven et al. 1996; Yi et al. 1996)rather than de novo synthesis of elements of the per-ception, the signal transduction pathways and of thetranscription machinery. Actually, even in the presenceof cycloheximide, CYP94A1 mRNAs accumulate in re-sponse to clofibrate (unpublished data), reinforcing thehypothesis of the involvement of activation reactions forits transcriptional regulation.

The high chemicals concentrations applied would beperceived as toxic considering the morphological anddevelopmental modifications of the V. sativa seedlingsthat occured in response to these treatments. Therefore,CYP94A1, in contrary to CYP94A2 and CYP94A3,

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should be considered as sensitive to chemical stresses. Itsinduction by SA (as reported above) and by methylj-asmonate (Pinot et al. 1998) suggests the involvement ofCYP94A1 also in response to biotic stresses. This lastpoint will be next investigated after treatment of the V.sativa seedlings by yeast elicitor.

The question arises why CYP94A1 expression is sodrastically and rapidly increased under these deleteriousconditions? The functional significance of the produc-tion of oxygenated FAs might be to synthesize mono-mers of plant protective polymers as cutin and suberin,to produce secondary signals of abiotic and bioticstresses and to detoxify free FAs. As mentioned above,CYP94A1, even necessary, does not constitute a limitingstep in the stem epidermal cutin biosynthesis. Its sig-nificance in root endodermis suberin biosynthesis shouldfurther be investigated by in situ hybridization studies bycomparison of its local expression in control and clofi-brate-treated seedlings.

Several indirect arguments are in favor of the impli-cation of oxidized FAs in plant defense reactions:Schweizer et al. (1996a, b) clearly demonstrated withdifferent models that pathogen-challenged plants per-ceived oxygenated FAs as endogenous molecules for theinduction of resistance. It is striking that 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids,which are produced in vitro with high efficiency byCYP94A1, also have the strongest effect in eliciting de-fense mechanisms. In addition, SA and methyl-jasmo-nate, which mediate pathogen attack, are stronginducers of CYP94A1. Another example of rapid chan-ges in FA metabolism in response to pathogen attackhas been described by Kirsch et al. (1997). According tothese authors, expression of a plastid-localized x-3 FAdesaturase was induced at fungal infection sites of Pet-roselinum crispum cells. Production of polyunsaturatedFAs as precursors in the generation of jasmonate hasbeen proposed by these authors to explain the biologicalrole of this enzyme.

Noteworthy, the profile of CYP94A1 regulation, bythe nature of the inducers and the concentrations neededfor effective induction, appears to be similar to thatdescribed for some plant GSTs. Over the last decade,several reports described the transcriptional activationof GSTs by pathogen attack and by a large variety ofchemical compounds, including SA, active and inactiveauxins, jasmonate, heavy metals, hydrogen peroxide,with the highest response elicited by elevated concen-trations of the inducing agent (Ellis et al. 1993; Ulmasovet al. 1994, 1995; Chen and Singh 1999). Recently, wemeasured a strong (12-fold) transcription activation ofone isoform of glutathione-S-transferase (GST6) in A.thaliana seedlings treated for 6 h by 200 lM and500 lM clofibrate solutions (unpublished data). Func-tional analysis of the GST promoter evidenced theimportance of an activation sequence-1 (as-1) cis-ele-ment, which confers transcription activation in responseto SA and active and inactive auxins (Ulmasov et al.1994, 1995).

In an effort to clarify the mechanisms of CYP94A1regulation, we determined its promoter sequence: inter-estingly, the 3 kb analyzed sequence contains a putativeas-1-like regulatory element (unpublished data). Func-tional analyses are now underway to investigate theimportance of this potential enhancer in the transcrip-tional regulation of CYP94A1. Our finding and theongoing analysis should provide more informationregarding the biological role of this plant FAs x-hydroxylase. The as-1-element occurs frequently in virus(e.g., cauliflower mosaic virus 35 S promoter) andAgrobacterium T-DNA genes (e.g., octopine and nopa-line synthase promoters), but rarely in plant cellulargenes, except some GSTs (Ellis et al. 1993). GSTsdetoxify a wide range of chemicals and may protectplant cells from reactive hydroperoxides produced as aconsequence of oxidative stress and defense responses.The evolutionary conservation of the as-1 element in thepromoter of CYP94A1 as in some GST promoters mightindicate their involvement in a common physiologicalprocess. Therefore, CYP94A1, nearly regulated as someGSTs by auxins, analogs, and SA, could also be involvedin detoxification reactions.

One described early auxin action is the activation ofphospholipase A2, which generates a rapid release offree FAs (Paul et al. 1998). These authors described theaccumulation of lysophospholipids (as a measure ofphospholipase A activity) in parsley and soybean cells assoon as 1–2 min after auxin application, but only atconcentrations well above 100 lM IAA. Free FAs thatare toxic for the cells must be rapidly converted to lesstoxic metabolites. In this context, induction ofCYP94A1, which has a broad substrate range for FAs,may constitute the first step in free FA catabolism. Thepossibility that activation of lipase followed by a releaseof free FAs would trigger CYP94A1 transcription inchemical-treated V. sativa seedlings may also be pro-posed by analogy with the mechanisms of transcrip-tional gene induction of FAs x-hydroxylases in animalsby the very same chemicals.

Production of activated oxygen species constitutesanother described early event in plant defense reactions,and induction of GST should be implicated in the releaseof the oxidative burst.

In conclusion, the similarities between chemical-dependent gene activation of CYP94A1 and GSTs, thelatter enzyme known involved in detoxification, suggesta defense mechanism against high doses of exogenouscompounds. It is most likely that these chemicals mimican endogenous ligand with greater affinity for thereceptor leading to CYP94A1 induction. Future work isaimed at characterizing this ligand.

Acknowledgements The authors acknowledge Ms. Marie-FrancePoulet for skilful technical assistance and Dr. Dawn Little forcritical reading of the manuscript. We thank gratefully Dr. BernardGrausem for effective computer assistance. We thank also Dr.Christiane Garaud (CNRS-IBMP-Strasbourg) for offering theopportunity for transmission electron microscopic investigations.We are indebted to Professor Felix Mauch (University of Fribourg,

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Switzerland) for the generous gift of an Arabidopsis GST6 cDNAclone. Financial support by the Centre National de la RechercheScientifique is gratefully acknowledged.

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