Plant Molecular Biology 36: 473–478, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme
from the California poppy Eschscholzia californica
Kay Hauschild, Hubert H. Pauli & Toni M. Kutchan
ur Molekulare Biologie, Universit
unchen, Karlstrasse 29, 80333 M
unchen, Germany (
Received 4 June 1997; accepted in revised form 30 September 1997
Key words: species-speciﬁc promoter, benzophenanthridine, alkaloids, Papaveraceae
A genomic clone, bbe1 was isolated that encodes the methyl jasmonate-inducible berberine bridge enzyme of
DNA gel blot analysis indicates that two genes are present in the E. californica genome that code for the berberine
bridge enzyme reading frame. Each coding region is apparently preceeded by a unique promoter sequence. The
bbe1 gene contains no introns and one transcriptional start site. A 41 nucleotide region between
of the 5
-ﬂanking region appears to be essential for promoter activity in E. californica. The promoter displayed an
unexpectedlyhigh species speciﬁcity, being active in only E. californica and Thalictrum bulgaricum, out of 28 cell
suspension cultures tested.
The benzophenanthridine alkaloids are antimicrobi-
al compounds that are biosynthetically derived from
L-tyrosine in various members of the Papaveraceae.
When species such as Eschscholzia californica,the
California poppy, or Papaver somniferum, the opium
poppy, are taken into cell culture, this class of alkal-
oids is elicitor-inducible [3, 10]. The entire biosyn-
thetic pathway from two molecules of L-tyrosine to
the most highly oxidized benzophenanthridine alkal-
oid macarpine has been elucidated at the enzyme
level (reviewed in ). Along this pathway, the eight
membrane-associatedenzymes were found to be indu-
cible by a variety of elicitor substances, including
members of the jasmonate class of compounds [6,
7]. The berberine bridge enzyme is one of these
membrane-associated, inducible enzymes [9, 11]. In
order to begin to understand the regulation of alkal-
oid biosynthesis in plants, we have initiated an ana-
lysis of the cis elements of the inducible promoters
of benzophenanthridine alkaloid biosynthetic genes.
Thenucleotidesequence data reportedwill appear in theEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AF005655.
The ultimate long-termed goals of this study are the
identiﬁcation of trans factors involved in the jas-
monic acid/octadecanoid-mediated induced of alkal-
oid biosynthetic genes. The unexpected result presen-
ted herein is the inherent difﬁculty of performing this
type of study with species of medicinal plants that are
less well moleculargeneticallycharacterizedthanAra-
bidopsis, tobacco, petunia and foodstuff plants such as
the cereal crops.
The berberine bridge enzyme gene bbe1 was isol-
ated from a genomic library prepared by ligation
of Sau3A restriction-digested E. californica genom-
ic DNA into BamHI-digested
Charon 40. The
labelled cDNA clone from this same species was
used as hybridization probe . Screening of 500000
plaques resulted in the isolation of one positive clone.
This positive clone was digested with the restriction
endonuclease XbaI, the fragments were resolved by
0.8% agarose gel electrophoresis and capillary blot-
ted onto a nylon membrane. Hybridization to the
P-labelled cDNA clone indicated that three bands
(4400, 2100 and 360 bp) contained bbe1 reading