Plant Molecular Biology 39: 1079–1090, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Novel characteristics and regulation of a divergent cinnamate
4-hydroxylase (CYP73A15) from French bean: engineering expression in
, Kristopher A. Blee
, Michel Schalk
, Daniele Werck-
D´epartement de Biologie Mol´eculaire des Plantes, CNRS-IBMP, 28 rue Goethe, 67083 Strasbourg Cedex,
Institute of Cytology and Genetics, 630090 Novosibirsk-90, Russia;
Department of Biochemistry,
Royal Holloway and Bedford New College, University of London, Egham, Surrey, TW20 0EX, UK (
Received 20 April 1998; accepted in revised form 6 October 1998
Key words: French bean, Phaseolus vulgaris L., differentiation, cytochrome P450, cinnamate 4-hydroxylase,
cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other
species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured
cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5
and a 140 bp 3
-untranslated region. The open reading frame, determined by sequence similarity, codes for a
protein with a predicted M
of 59 229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found
in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and
C-terminus and in four domains internally, so that overall sequence similarity is in the range 58–66%. Many clones
contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is
sufﬁcient for it to be classiﬁed as CYP73A15, although it is the least similar member of this family classiﬁed so far.
The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal
of the intron. High-level expression also required modiﬁcation of the N-terminus to that of CYP73A1. Yeast did
not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA
for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean
but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells
undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-
hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the
majority of CYP73As cloned so far are involved.
Abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; CCR, cinnamoyl CoA
reductase; CAD, cinnamyl alcohol dehydrogenase.
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers Y09447, Y09448 and Y09449.