Plant Molecular Biology37: 39–52, 1998. 39c 1998Kluwer Academic Publishers. Printed in Belgium.

cytochrome p450 superfamily inArabidopsis thaliana: isolation of cDNAs,Differential Expression, and RFLP mapping of multiple cytochromes P450

Masaharu Mizutani1, Eric Ward2 and Daisaku Ohta3;�

1International Research Laboratories, Ciba-Geigy Japan Ltd., 10-66 Miyuki-cho, Takarazuka 665, Japan;2Biotechnology and Genomics Center, Novartis Crop Protection, Inc., 3054 Cornwallis Road, Research TrianglePark, NC 27709-2257, USA;3Research Institute for Biological Sciences, Okayama 7549-1 Yoshikawa, Kayo-cho,Okayama 716-12, Japan; (�author for correspondence)

Received 1 July 1997; accepted in revised form 29 November 1997

Key words: Arabidopsis, cytochrome P450, RFLP mapping, wounding, circadian rhythm

Abstract

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) fromArabidopsis thalianaemploying aPCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved betweentwo plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found inthe N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-oldArabidopsisas a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAsencoding different P450s have been isolated from anArabidopsiscDNA library. These P450s have been assignedto either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C,CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that theregions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s.The N-terminal structures of the predicted P450 proteins suggested that theseArabidopsisP450s were located atthe endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One ofthe clones,CYP71B2, was located at a position very close to thega4andgai mutations. RNA blot analysis showedexpression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding andlight/dark cycle, implicating involvement of these P450s in diverse metabolic processes.

Introduction

Cytochrome P450 (P450) is a generic name of a familyof heme-thiolate proteins involved in oxidative meta-bolism of diverse endogenous and exogenous lipophil-ic substrates. Up to now, more than 400 P450 geneshave been identified from a wide range of organismsfrom bacteria to animals [34].

In higher plants, P450s also play crucial roles inbiosynthesis of a variety of endogenous lipophilic com-pounds such as fatty acids, sterols, phenylpropanoids,

The nucleotide sequence data reported will appear in theEMBL, GenBank and DDBJ Nucleotide Sequence Databases underthe following accession numbers: CYP71B2, D78605; CYP71B3,D78602; CYP71B4, D78603; CYP71B5, D78601; CYP71B6,D78604; CYP76C1, D78600; CYP83A1, D78599; CYP83B1,D78598; CYP91A1, D78606; CYP91A2, D78607.

terpenoids, phytoalexins, and gibberellins [4, 10]. Inaddition, oxidative detoxification of a number of herb-icides in plant tissues is also achieved by a P450-dependent monooxygenase system [10, 18, 39, 40].Despite these important roles, studies of plant P450shave been impeded by the difficulties in purificationof P450s due to their instability and low abundancein tissues. So far, only a limited number of P450enzymes and their corresponding cDNAs have beenisolated from plants [22, 23, 30, 37, 38, 41, 45]. Non-etheless, the observation of various P450-dependentreactionsin vitro suggested the existence of multiplediverse P450s in a single plant species [4, 10].

Recent progress in molecular biology has facilit-ated the isolation of novel P450 clones from plants.Several cDNAs belonging to three P450 families were

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isolated from eggplant using an eggplant CYP75 cDNAas a probe [47, 48], and four P450 cDNAs of theCYP71A subfamily have also been cloned from maize[14]. Another successful approach is based on PCRusing primers designed from conserved amino acidsequence around the heme-binding domain of P450proteins. This strategy is particularly useful to isol-ate P450 cDNAs of low abundance or unstabile P450.Holton et al. [19], employing this approach, isolated18 P450 cDNA fragments fromPetunia and foundthat two clones encoded P450s with flavonoid 30

; 50-hydroxylase activity. It has also been reported that 15cDNA fragments were isolated from periwinkle witha PCR approach [28], and 4 P450 cDNAs were isol-ated from pea using reverse transcription-PCR [13].However, it is usually difficult to elucidate physiolo-gical functions of the P450s isolated through thesemolecular biological approaches.

In this paper, we report the isolation of 13 full-length and 3 partial P450 cDNAs fromArabidopsisthaliana. Eleven P450s have been assigned to 5 distinctfamilies, CYP71B, CYP73A, CYP76C, CYP83, andCYP91. The N-terminal structures of the deduced pro-teins have shown properties similar to those of mam-malian microsomal P450 proteins, suggesting micro-somal localization of theseArabidopsisP450s. RNAblot analysis revealed tissue characteristic expressionpatterns for each of the P450s, as well as differentresponsiveness to wounding and light. One of the isol-ated P450s, CYP73A5, has been already identified ascinnamate 4-hydroxylase [31], whereas physiologicalfunctions of the other 15 P450s are still unknown. As afirst step towards the elucidation of their physiologicalfunctions, genetic loci of four P450 genes have beendetermined by RFLP mapping, and a linkage map withthe loci of known mutations is reported.

Materials and methods

Plant materials/treatments

Arabidopsis thalianaecotype Columbia (Col-0) (LehleSeeds, Tucson, AZ) seedlings were grown under sterileconditions on 0.8% (w/v)agarose plates containing GMmedium [49] in a growth chamber maintained at 22�Cunder continuous light or a light/dark cycle (9/15 h).

For wounding treatment, fully developed leavesfrom 3-week-old plants grown under continuous lightwere cut into segments 2 mm in width and incubatedfor 1 to 9 h under continuous light in a Petri dish con-

taining GM medium and 50�g/l chloramphenicol. Forthe 0 h time point, sliced leaves were frozen immedi-ately without incubation.

Polymerase chain reaction

Total RNA was prepared from 7-day-old seedlingsby phenol/chloroform extraction followed by lithi-um chloride precipitation as described by Lagriminiet al. [24] and poly(A)+RNA was isolated from thetotal RNA using a poly(A)+ Quick mRNA isolationkit (Stratagene, La Jolla, CA). First-strand cDNAwas synthesized from 1�g of the poly(A)+ RNAusing Moloney Murine Leukemia Virus reverse tran-scriptase (Toyobo, Osaka, Japan) and was used as atemplate for PCR with several sets of primers (Fig-ures 1, 2) designed from the sequences conservedbetween CYP71A1 and CYP73A2. The PCR was car-ried out in a reaction mixture (100�l) consisting of10 mM Tris-HCl (pH 8.3), degenerate primers (at10�M each), 200�M dATP, 200�M dCTP, 200�MdTTP, 200�M dGTP, 2 mM MgCl2, 50 mM KCland 25 units/mL AmpliTaq DNA polymerase (PerkinElmer/Cetus, Norwalk, CT). The reactions were per-formed through 35 cycles of 30 s at 94�C, 30 s at50�C and 90 s at 72�C using a thermal cycler (PerkinElmer/Cetus, model 480). PCR products were sep-arated by low-melting-temperature agarose gel (1.5%w/v) electrophoresis and were cloned into the pCRIIvector using a TA cloning kit (Invitrogen). The clonedPCR products were divided into several groups by ana-lyzing their restriction fragments after digestion withEcoRI, XhoI, andHindIII. The DNA sequences of thecloned PCR products were partially determined.

Isolation of cDNA clones

Each of the PCR products was labeled with [32P]-dCTPby the random priming labelling method [12].A total of600 000 phage plaques from anArabidopsiscDNA lib-rary [31] was screened for corresponding P450 cDNAsunder high stringency conditions: hybridization, 16 hat 65�C in a hybridization buffer containing 1% (w/v)BSA, 7% (w/v) SDS, 50 mM sodium phosphate pH 7.5,and 1 mM EDTA [9] washing, 10 min in 2�SSC, 0.1%(w/v) SDS at room temperature, and 20 min at 65�Cin 0.1� SSC plus 0.1% (w/v) SDS.

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Figure 1. Comparison of the deduced amino acid sequences between CYP71A1 [5] and CYP73A2 [30]. Identical amino acid residuesare shadowed with gray, and dashs are inserted to maximize the sequence homology. The amino acid sequences used to design degenerateoligonucleotide primers are underlined.

DNA preparation and DNA blot analysis

Genomic DNA was isolated from shoots of 3-week-old Arabidopsisseedlings and purified by ethidiumbromide-cesium chloride density gradient centrifuga-tion as described previously [1]. For Southern blots,genomic DNA was digested with restriction enzymes,separated by 0.7% (w/v) agarose gel electrophores-is, and transferred onto nylon membranes. The isol-ated full-length P450 cDNAs were used as probes, andhybridization and washing were carried out under thestringency conditions described above.

RFLP mapping was performed by L. Medrano inthe laboratory of E. Meyerowitz as described by Changet al. [6] using genomic DNA isolated from a crossbetween Landsbergerecta and Columbia ecotypes.Gel blots of genomic DNAs digested withXbaI were

probed with the P450 cDNAs to reveal DNA poly-morphisms between Landsberg and Columbia. RFLPmapping data were analyzed using the MAPMAKERcomputer program developed by Landeret al. [25].

RNA preparation and RNA blot analysis

Total RNA was isolated by phenol/chloroform extrac-tion followed by lithium chloride precipitation asdescribed by Lagriminiet al. [24]. Samples oftotal RNA (5 �g) were separated by 1.2% (w/v)formaldehyde-agarose gel electrophoresis, and blottedonto a nylon membrane (GeneScreen Plus, New Eng-land Nuclear, Boston, MA). Hybridization and wash-ing were carried out under the high-stringency condi-tion described above. The hybridization signals werequantified using an imaging analyzer BAS2000 (Fuji

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Photo Film Co., Kanagawa, Japan). The hydridizedDNA probes were stripped from the membranes byboiling in a buffer containing 0.01� SSC and 0.01%(w/v) SDS for 20 min, and blots were re-hybridized toan Actin-1 probe [32] for normalization.

DNA sequencing and analysis

DNA sequencing was performed using a DyeDeoxyTerminator Cycle Sequencing kit (Applied Biosys-tems, CA) and an automated DNA sequencer (AppliedBiosystems 373A). Sequence analysis was performedusing DNASIS Ver.3.5 software (Hitachi SoftwareEngineering America, San Bruno, CA).

The sequences of the newly isolated P450 cDNAswere submitted to the P450 Nomenclature Committeefor the assignment of P450 family names [33, 34].

A P450 protein sequence from one gene familyusually is defined as having�40% amino acid iden-tity to a P450 protein from any other family. Naminga P450 gene includes the root symbol CYP, denotingcytochrome P450, an Arabic number designating theP450 family, a letter indicating the subfamily when twoor more subfamilies are know to exist within that genefamily, and an Arabic numeral representing the indi-vidual gene. The same numbers and letters are recom-mended for the corresponding gene product (mRNA,cDNA, enzyme) [33, 34].

Results

PCR strategy

We isolated novel P450 clones from 7-day-oldAra-bidopsisseedlings by a PCR strategy. While more than200 P450 genes from a variety of organisms had beenreported [33] when we started this study, only twoplant P450s were available: avocado CYP71A1 of anunknown physiological function [5] and a mung beancinnamate 4-hydroxylase, CYP73A2 [30]. CYP71A1and CYP73A2 were 30% identical at the amino acidlevel, and we were able to design degenerate oligonuc-leotide primers from five conserved regions as shown inFigure 2. First, the consensus sequence for the heme-binding region (HR2 region) [15] was identified asPFG(A/V)GRR in CYP71A1 and CYP73A2 (Figure 1,underlined), and a primer(MA39) was designed fromthis plant HR2 region (Figure 2). Another primer (Fig-ure 2, MA47) was designed from a proline-rich motif(KLPPGP) found next to the N-terminal signal-anchor

Figure 2. Degenerate oligonucleotide primers for PCR. Degener-ate oligonucleotide primers were designed from five amino acidsequences conserved between CYP71A1 and CYP73A2 (Figure 1,underlined).

sequence CYP73A2 (Figure 1, underlined) [42, 43].A proline-rich motif, (P/I)PGPX(P/G)XP, is known tobe conserved in mammalian microsomal P450s [51].Next, we found that a short sequence, YGE(H/Y)WR,is conserved in the middle region of CYP71A1 andCYP73A2 (Figure 1, underlined), and a forward primerwas designed from this sequence (Figure 2, MA46). Areverse primer (Figure 2, MA48) was designed froma relatively long sequence, (A/P)EEF(R/L)PERF, con-served in the C-terminal region of both CYP71A1 andCYP73A2. Finally, a sequence, NAWAIGRDP, fromthe C-terminal region of CYP71A1, was chosen todesign a primer (MA40, Figure 2). Since only 4 out ofthe 9 residues were consistent with the correspondingregion of cinnamate 4-hydroxylase, CYP73A2 (Fig-ure 1, underlined), it was our intent to obtain P450clones other than CYP73A2. We wanted to avoid theamplification of CYP73A2, which is one of the mostabundant P450s in plants and whose mRNA should

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Table 1. Putative P450 cDNA fragments amplified by PCR. PCR wasperformed with six sets of the degenerated primers (Figure 2) using a first-strand cDNA derived from a 7-day-oldArabidopsisseedlings as a templateand the PCR products were cloned into a pCRII vector with a TA cloningkit.

Set of primers Amplified P450 fragments

MA46 �MA39 pCR-C4H-a pCR-12-a pCR-67-a

MA46 �MA40 pCR-65 pCR-66 pCR-67-b

MA46 �MA48 pCR-C4H-b pCR-12-b pCR-48

MA47 �MA39 pCR-C4H-c pCR-4-a pCR-12-c

MA47 �MA40 pCR-2 pCR-3 pCR-4-b pCR-13

MA47 �MA48 pCR-C4H-d pCR-23

therefore constitute a major part of plant P450 tran-scripts.

Using six primer sets (Table 1), we performedPCR on first strand cDNA prepared from a poly(A)+

RNA of 7-day-oldArabidopsisseedlings as a tem-plate, and 18 different fragments were amplified, cor-responding to 11 distinct P450s (Table 1). Sequenceanalysis revealed that 4 clones, designated P450C4H(Table 1), were derived from CYP73A5, cinnamate 4-hydroxylase, fromArabidopsis[31]. The remaining 14PCR products appeared to encode 10 novel P450s.Fourclasses of clones were obtained with the MA39 primerdesigned from the heme-binding region. On the otherhand, these classes as well as 7 more were amplifiedusing the four primers other than MA39, indicatingthat various regions homologous between CYP71A1and CYP73A2 are conserved in other plant P450s.

Isolation of cDNA clones

We screened a cDNA library from 7-day-oldArabidop-sisseedlings using the PCR fragments as hybridizationprobes under high stringency conditions and obtainedcorresponding cDNAs (Table 2). Some of the probeshybridized to multiple cDNAs: pCR-13 hydridized to acorresponding cDNA (P450-13-6) and three additionaldifferent cDNAs (P450-13-1, 13-5 and 13-7); pCR-66hybridized to its corresponding cDNA (P450-66-8) andan additional one, P450-66-5; pCR-67 hybridized toits corresponding P450-67-3 and another cDNA clone,P450-67-1. The remaining PCR clones hybridized onlyto their corresponding cDNAs. DNA sequences of 11of the 16 cDNAs were completely determined, andthe deduced primary structures are shown in Figure 3.These P450s were assigned to five distinct gene fam-ilies (see below and Table 2). Sequence comparison(data not shown) indicated that P450-65 cDNA lacked

Table 2. P450 cDNA clones isolated using the cor-responding PCR clones as probes.

PCR clones P450 cDNA clones Family

pCR-C4H-a P450-C4H CYP73A5

pCR-2 P450-2 CYP83B1

pCR-3 P450-3 CYP83A1

pCR-4-a P450-4 CYP76C1

pCR-12-a P450-12 CYP71B5

pCR-13 P450-13-1 CYP71B3

P450-13-5 ND

P450-13-6 CYP71B4

P450-13-7 ND

pCR-23 P450-23 CYP71B6

pCR-48 P450-48 CYP71B2

pCR-65 P450-65 CYP91A1

pCR-66 P450-66-5 ND

P450-66-8 CYP91A2

pCR-67-a P450-67-1 ND

P450-67-3 ND

ND: Not determined.

a few bases at the 50 terminus of a corresponding full-length cDNA. Partial DNA sequencing showed that theremaining 5 clones, P450-66-5, P450-67-1, and P450-67-3 also represent full-length cDNAs and P450-13-5 and P450-13-7 are partial clones (data not shown).Thus, we have isolated 13 full-length and 3 partialcDNAs encoding 16 distinct P450s fromArabidopsis.

Multiple P450 gene families inArabidopsis

CYP71Five P450 cDNAs (P450-12, 13-1, 13-6, 23, and48) were 44–69% identical at the amino acid levelto one another (Table 3), and their deduced amino acidsequences were more than 40% identical to that of

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Figure 3. Amino acid sequence alignment for the 11 P450 cDNAs fromArabidopsis. The amino acid sequences deduced from the 11 P450cDNA sequences were aligned by the program, Clustal W [46]. Identical amino acid residues are shadowed with a gray color, and dashs areinserted to maximize the sequence homology. The proline-rich motif and the heme-binding domain (HR2 region [15]) are underlined. Aminoacid residues conserved among all P450s are indicated by asterisks under the alignment.

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avocado CYP71A1. These P450s were assigned to theCYP71B subfamily as follows: P450-12, CYP71B5;P450-13-1, CYP71B3; P450-13-6, CYP71B4; P450-23, CYP71B6; P450-48, CYP71B2. CYP71B3 andCYP71B4, isolated using the same probe (pCR-13),showed the highest identity to each other at both theamino acid (68.6%) and the DNA levels (76.7%).Twenty-two P450s belonging to the CYP71 familyhave so far been isolated from several plant species[34]. However, none of their physiological functionshave yet been identified. The high degree of diversityamong the predicted protein sequences of the Ara-bidopsis CYP71B P450s (Figure 3) suggests distinctcatalytic activities.

CYP73The P450-C4H cDNA (Table 2) was completelyidentical to the CYP73A5-encoding cDNA (cinnamate4-hydroxylase) previously isolated fromArabidopsis[31]. P450 clones belonging to CYP73 have also beenisolated from several plant species: CYP73A1 fromHelianthus tuberosus[45], CYP73A2 fromPhaseol-us aureus[30], CYP73A3 fromMedicago sativa[11],CYP73A4 fromCatharanthus roseus[20], CYP73A9from Pisum sativum[13], and CYP73A10 fromPet-roselinum crispum[27]. It has already been shownfrom heterologous expression experiments that fiveP450s belonging to the CYP73 family (CYP73A1through CYP73A5) have cinnamate 4-hydroxylaseactivity [11, 20, 45]. While the CYP73 P450s so farisolated are 75–90% similar to one another at the aminoacid level, it is not clear whether or not all the mem-bers of CYP73 catalyze the same reaction. It has beenreported that single amino acid substitutions in P450proteins can result in dramatic changes of substratespecificity [21, 26].

CYP76The deduced primary structure of P450-4 was 26 to37% homologous to those of the other ArabidopsisP450s (Table 3). The highest identity (40%) was foundwith CYP76A2, isolated fromSolanum melongenahypocotyls using a CYP75 cDNA as a probe [47].P450-4 was therefore assigned to a new P450 subfam-ily, CYP76C1. Four P450s in this family have beenisolated [34], but their physiological functions are stillunknown.

Figure 4. Genomic Southern blot analysis of 10 P450 genes ofArabidopsis. A 1�g portion ofArabidopsisColumbia genomic DNAwas digested with the restriction enzymes: B,BamHI; E, EcoRI; H,HindIII. The digested genomic DNA was separated on a 0.7% (w/v)agarose gel, blotted onto nylon membrane, and hybridized with[32P]-labeled full-length cDNAs of the 10 P450s indicated. Themigration of size markers is shown to the right of the blots.

CYP83 and CYP91Sequence comparison showed that P450-3 was com-pletely identical to CYP83A1 previously isolatedby cross-hybridization with a gene rescued from aT-DNA-tagged fah1 mutant of Arabidopsis, whichlacks ferulate 5-hydroxylase activity [7, 29]. P450-2 shares 61.6% identity at the amino acid level withCYP83A1 and was assigned to its own new subfamilyas CYP83B1.

P450-65 and P450-66-8, which are 50.4% identicalat the amino acid level (Table 3), have been assigned toa new P450 family, CYP91A1 and CYP91A2, respect-ively. No P450s belonging to this family have beenreported from other plant species.

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Table 3. Homology of DNA and amino acid sequences of the P450s isolated fromArabidopsis.

71B2 71B3 71B4 71B5 71B6 73A5 76C1 83A1 83B1 91A1 91A2

Nucleotide identity (%)

71B2 (P450-48) – 65.8 66.2 63.7 55.0 49.2 50.6 52.1 54.1 45.6 48.1

71B3 (P450-13-1) 57.3 – 76.1 60.8 50.6 49.5 49.9 48.7 49.8 47.2 48.8

71B4 (P450-13-6) 57.8 68.6 – 61.9 56.5 49.8 50.7 53.4 53.1 48.0 47.9

71B5 (P450-12) 54.1 54.1 51.0 – 54.6 49.6 48.9 51.0 52.9 44.1 46.0

71B6 (P450-23) 43.6 45.0 46.1 46.8 – 49.0 50.1 50.9 52.8 48.0 47.9

73A5 (P450-C4H) 26.2 28.9 32.0 29.3 27.9 – 49.0 50.4 49.8 46.2 47.8

76C1 (P450-4) 34.6 34.9 35.4 31.5 36.2 25.8 – 50.4 51.0 47.1 48.5

83A1 (P450-3) 36.9 37.3 37.7 30.8 36.0 29.9 33.0 – 63.5 46.6 50.0

83B1 (P450-2) 40.2 41.2 39.3 36.9 40.2 28.5 34.4 61.6 – 48.6 48.4

91A1 (P450-65) 33.5 33.3 34.8 32.1 31.9 28.2 32.0 29.7 30.0 – 57.6

91A2 (P450-66-8) 31.9 32.8 32.9 31.0 30.0 25.3 32.0 29.6 32.150.4 –

Amino acid identity� (%)

�Amino acid identities higher than 40% are shown in bold type.

Of 74 P450 families from both eukaryotes andprokaryotes, 23 families (CYP51 and CYP71 throughCYP92) are represented in higher plants [34]. TheP450s isolated in this study belong to either establishedsubfamilies; CYP71B, CYP73A and CYP83A, or nov-el subfamiles; CYP76C, CYP83B and CYP91A. Thus,a total of 10 families, including those so far reported(CYP72, CYP74, CYP84, CYP86 and CYP90) havebeen now established inArabidopsis.

Protein structuresThe P450s isolated in this study have significantly highhomology throughout their entire sequences including35 conserved amino acid residues (Figure 3). In addi-tion, each P450 contains a hydrophobic stretch at itsN-terminus (Figure 3), which has the structural prop-erties of the signal-anchor sequence of microsomalP450s [35]. The region contains a few charged residuesand a proline rich motif following the hydrophobicstretch, both of which are responsible for the target-ing and anchoring of newly synthesized P450 proteinsat the cytoplasmic surface of the endoplasmic retic-ulum [2, 51]. An extended consensus sequence of thecore heme-binding site, FxxGxR/HxCxG, was iden-tified as P/AFGxGRR/KxCPG/A in these Arabidop-sis P450s (Figure 3, underlined). Interestingly, thehighly conserved glycine found 2 residues C-terminalto the heme-ligating cysteine was replaced by alan-ine in CYP71B6 and CYP83B1. Of more than 400P450 sequences so far determined, this substitutionhas been found in only 10 P450s from insect, fungi,and plants [34]. It is not known how the substitution of

this structurally important glycine influences the activesite conformation.

Genomic Southern blot analysis

We have previously reported that CYP73A5 is encodedby a single-copy gene inArabidopsis[31]. To estim-ate the copy number of the other 10 P450 genesdescribed here, we performed genomic Southern blotanalyses under high stringency (65�C in 0.1� SSCand 0.1% SDS). We observed distinct hybridizationpatterns unique to each P450 gene (Figure 4), whichindicated that the individual hybridization probes didnot cross-hybridize to other P450 genes. The hybridiz-ation patterns in the genomic Southern analysis (Fig-ure 4) were consistent with those predicted from therestriction patterns of the corresponding cDNAs (datanot shown), suggesting that each P450 is encoded by aunique single copy gene inArabidopsis.

RFLP mapping of the four P450 genes

In an attempt to study whether the isolated P450 genesare closely linked to known mutations, the genetic mappositions of the P450 genes were determined by RFLPmapping [6]. CYP71B2, CYP71B6, CYP83A1, andCYP91A1exhibitedXbaI polymorphism between theColumbia and Landsberg ecotypes (data not shown).These 4 genes were located on different chromosomesas shown in Figure 5.

CYP71B2andCYP71B6mapped to chromosome 1and 2, respectively.CYP91A1mapped to chromosome

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Figure 5. RFLP mapping of theCYP71B2, CYP71B6, CYP83A1, andCYP91A1genes. RFLP markers and the four P450 genes are shown onthe schematicArabidopsisgenetic map. The numbers at left indicate the map distance (in cM) between markers.

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5, and its map position (between CD06455f and phyC)is near thega3 locus (between m291 and CD06455f),which is involved in a P450-dependent oxidation ofkaurene in gibberellin biosynthesis [52]. Another P450gene,CYP71B2mapped 0.2 cM below an RFLP mark-er, m241, on chromosome 1. It should be noted thatthe ga4, gai, anddw1 mutations have also been loc-alized ca. 0.6 cM, 0.8 cM and 1.2 cM distant fromm241, respectively [17]. These results raise the pos-sibility that genes involved in gibberellin metabolismmight exist as a cluster. Chianget al. [8] have recentlydemonstrated that theGA4gene encodes a gibberellin3�-hydroxylase in the gibberellin biosynthetic path-way of Arabidopsis. Among biochemical pathways inplants, the biosynthesis of gibberellins is thought toinvolve several P450 enzymes [16]. However, involve-ment ofCYP71B2in gibberellin metabolism remainsto be clarified by genetic or biochemical means.

Expression patterns inArabidopsis

Northern blot analysis was performed to investigate theexpression of these 11 P450s in Arabidopsis using thefull-length cDNAs as probes. These P450 genes weredifferentially expressed in roots, leaves, inflorescencestems, flowers, and siliques as shown in Figure 6A. TheCYP71B3andCYP71B4genes were highly expressedin leaves but not in the other organs. TheCYP71B2transcript was detected in roots, leaves, and stemsbut not in flowers and siliques. The expression levelsof CYP71B5and CYP71B6were highest in leaves,and CYP71B5mRNA was not detectable in roots.CYP73A5expression was highest in stems and high-er in roots and siliques than in leaves and flowers.CYP76C1was expressed at a higher level in flowersthan in the other organs and not expressed in roots.The CYP83A1expression level was highest in leavesand was significantly higher in roots and stems thanin flowers and siliques. In contrast to relatively lowexpression levels of the other P450 genes in roots, theCYP83B1gene was most strongly expressed in roots.TheCYP91A1mRNA level was highest in leaves com-pared with that in the other organs, whileCYP91A2mRNA was strongly expressed in flowers. The resultsdemonstrated that each of the P450 genes, even with-in the same P450 gene subfamilies, showed uniqueexpression patterns.

Figure 6B shows age-dependent expression pat-terns of the P450 genes. TheCYP71B2, CYP73A5,and CYP76C1genes were expressed at almost con-stant levels throughout leaf aging, while the other P450

Figure 6. Tissue specific expression of the 11 P450 genes inAra-bidopsis. A. Total RNA was isolated from the roots and leaves of3-week-old plants, from inflorescence stems and flowers of 4-week-old plants, and from the siliques of 5-week-old plants. B. TotalRNA was isolated from the leaves of different age from 3-week-oldplants with 12 leaves. The first and second leaves represented theolder leaves. The middle-aged leaves were collected from the fourthand fifth positions, and the younger leaves were from the ninth andtenth positions counted from the bottom. Plants were grown undercontinuous light. A 5�g portion of total RNA was separated onformaldehyde agarose gels, transferred onto nylon membranes, andhybridized to the indicated probes.

genes were more strongly expressed in older leavesthan in younger leaves. Specifically, the expressionlevels ofCYP71B3, CYP71B4, CYP83A1, CYP91A1,andCYP91A2in older leaves were 4-,7-, 9-, 5-, and 13-fold higher, respectively, than those in younger leaves.

The expression of the P450 genes was stronglyaffected by wounding (Figure 7A). The strong induc-tion of CYP73A5 (cinnamate 4-hydroxylase) bywounding was consistent with our previous observa-tion which showed a coordinated induction of phenyl-propanoid pathway genes by wounding [31]. Theexpression levels ofCYP71B3, CYP71B6, CYP83B1,

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Figure 7. Effect of wounding and light/dark cycle on expressionlevels of the 11 P450 genes. A. Leaves were havested from 3-week-old plants grown under continuous light. The harvested samples werecut into strips 2 mm in width, and incubated for 9 h under continuouslight in a Petri dish containing GM medium. Total RNA was isolatedat the times indicated after wounding and analysed by RNA gelblotting (5�g per lane) using the probes indicated. B. Plants weregrown under a 9 h light/15 h dark cycle: light period 00:00–09:00;dark period 09:00–24:00. Total RNA was isolated from the leaves of3-week-old plants at the times indicated after the onset of the lightperiod (0 h), and analysed by RNA gel blotting (5�g per lane) usingthe probes indicated.

CYP91A1, and CYP91A2also increased 2-, 4-, 5-, 7-, and 2-fold, respectively, during 9h of thewounding treatment. In contrast, mRNA levels ofCYP71B2, CYP71B4, CYP71B5, CYP76C1, andCYP83A1decreased after wounding.

Expression patterns of the P450 genes during adiurnal light/dark cycle (9/15 h) were also investig-ated (Figure 7B). The transient induction ofCYP73A5expression within 3h in the light was consistent withour previous observation [31]. The expression of

CYP71B4, CYP76C1, andCYP83A1increased 3-, 5-,and 10-fold, respectively, during the light period anddecreased to a basal level within 3 h in the dark. ThemRNA level ofCYP71B2andCYP71B3decreased dur-ing the light, and the expression of the other P450 genesdid not change significantly during light/dark cycle.

Discussion

We have isolated 16 distinct P450 cDNAs fromAra-bidopsis. Presumably, there are a large number of addi-tional unidentified P450 genes inArabidopsis. Indeed,many unknown P450 clones have been deposited in theArabidopsisEST database [36], and several of theseEST clones are highly homologous to the P450s repor-ted here (data not shown). Furthermore, it is possiblethat we might have missed many P450 clones inAra-bidopsisin this experiment, either because of the PCRprimers we used, or because we screened only a singlecDNA library prepared from 7-day-oldArabidopsisunder high stringency conditions. Many P450 genesare thought to be spatially and temporally regulated indifferent manners. Novel P450s could be isolated fromcDNA libraries prepared from either different organsof different age or from tissues treated by wounding,pathogen infection, or UV irradiation. So far, 23 P450gene families including those 10 found inArabidopsishave been found in higher plants [34]. The number ofcloned P450 genes will increase through approachessuch as PCR-based strategies,ArabidopsisEST database searches, andArabidopsisgenome sequencing;however, the physiological functions of the identi-fied P450s will be difficult to elucidate through theseefforts.

Expression patterns of the P450 genesin plantawill in some cases have physiological implications.For example,CYP73A5(C4H) is well known to becoordinately induced by light with the genes involvedin the phenylpropanoid/flavonoid pathway such asphenylalanine ammonia-lyase [4, 27, 31]. In this study,we found that several P450s other thanCYP73A5werealso induced by light (Figure 7B). It has been repor-ted that a pigmentation/flowering-relatedP450, flavon-oid 3050-hydroxylase (CYP75), is induced by light [4,19]. Interestingly,CYP76C1, which was most stronglyinduced by light, was predominantly expressed inflowers (Figure 7B).

Wounding treatment also resulted in strong induc-tion of specific P450 species, while some of them weresignificantly downregulated (Figure 7A). Namely,

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CYP71B3, CYP71B6, CYP83B1, CYP91A1 andCYP91A2expression levels were higher in older leavesthan in younger leaves, and these P450s were inducedby wounding. It has been reported thatCYP71A1was induced during increased ethylene biosynthesistriggered by wounding [4]. P450s are also involved indefense mechanisms in plants, which include wound-healing, protection against UV irradiation, resistanceagainst pathogen infection and metabolism of xenobi-otics. For instance, it has been reported that the levelsof several P450s: C4H, isoflavone 20-hydroxylase, andisoflavone synthase in alfalfa suspension cells and 6�-hydroxylase in soybean were enhanced by elicitor chal-lenge, leading to the biosynthesis of the isoflavonoidphytoalexins [4]. It remains to be investigated howthese newly isolated P450s in Arabidopsis will respondunder various stress conditions.

Novel P450 clones are interesting candidates forheterologous expression, and the expressed recombin-ant P450s can be characterized in terms of their cata-lytic activities. However, even if a reaction catalyzedby such a recombinant P450 can be identified, it is notnecessarily a principal physiological role of the P450.In other words, it is possible that the identified reac-tion should be ascribed to another P450in vivo, andthe P450 of interest may have a completely differentphysiological function. An interesting approach is touse P450s of unknown function to generate sense- andantisense-expressing transgenic plants. Also, genet-ic linkage may be used to explore functions of newP450s. Recently, several P450 clones have been isol-ated from mutated plants by either T-DNA tagging ortransposon tagging, and functions of these P450s havebeen investigated [3, 29, 44, 50]. In this paper, wedetermined the loci of four P450 genes by RFLP map-ping, and it appears that one of them might be involvedin a metabolic step related to gibberellins.

P450s of identified catalytic function representpromising candidates for manipulating specific planttraits.

Acknowledgments

We are deeply grateful to the late Emeritus ProfessorRyo Sato for his invaluable advice and discussion. Wealso gratefully acknowledge Sharon Potter for help-ful advice on cloning, Roseann Novitzsky for DNAsequence analysis, Nobuko Uodome for care of theplants,and Leonard Medrano and Eliot Meyerowitz forkindly carrying out the RFLP mapping analyses.

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