Plant Molecular Biology 37: 39–52, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
cytochrome p450 superfamily in Arabidopsis thaliana: isolation of cDNAs,
Differential Expression, and RFLP mapping of multiple cytochromes P450
and Daisaku Ohta
International Research Laboratories, Ciba-Geigy Japan Ltd., 10-66 Miyuki-cho, Takarazuka 665, Japan;
Biotechnology and Genomics Center, Novartis Crop Protection, Inc., 3054 Cornwallis Road, Research Triangle
Park, NC 27709-2257, USA;
Research 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
We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a
PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between
two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in
the N-terminal region, and 11 putative P450 fragments were ampliﬁed from ﬁrst-strand cDNA from 7-day-old
Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs
encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned
to 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 the
regions 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 these Arabidopsis P450s were located at
the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of
the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed
expression patterns unique to each of the P450s in terms of tissue speciﬁcity and responsiveness to wounding and
light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.
Cytochrome P450 (P450) is a generic name of a family
of heme-thiolate proteins involved in oxidative meta-
bolism of diverse endogenousand exogenous lipophil-
ic substrates. Up to now, more than 400 P450 genes
have been identiﬁed from a wide range of organisms
from bacteria to animals .
In higher plants, P450s also play crucial roles in
biosynthesisofa varietyof endogenouslipophiliccom-
pounds such as fatty acids, sterols, phenylpropanoids,
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the 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]. In
addition, 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 P450s
have been impeded by the difﬁculties in puriﬁcation
of P450s due to their instability and low abundance
in tissues. So far, only a limited number of P450
enzymes and their corresponding cDNAs have been
isolated from plants [22, 23, 30, 37, 38, 41, 45]. Non-
etheless, the observation of various P450-dependent
reactions in vitro suggested the existence of multiple
diverse 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