Plant Physiol, February 2002, Vol. 128, pp. 696-706
Antisense RNA-Mediated Suppression of Benzophenanthridine
Alkaloid Biosynthesis in Transgenic Cell Cultures of California
Poppy1
Sang-Un
Park,
Min
Yu,2 and
Peter J.
Facchini*
Department of Biological Sciences, University of Calgary, Calgary,
Alberta, Canada T2N 1N4
California poppy (Eschscholzia californica
Cham.) cell cultures produce several benzophenanthridine alkaloids,
such as sanguinarine, chelirubine, and macarpine, with potent
pharmacological activity. Antisense constructs of genes encoding
two enzymes involved in benzophenanthridine alkaloid biosynthesis,
the berberine bridge enzyme (BBE) and N-methylcoclaurine
3'-hydroxylase (CYP80B1), were introduced separately into California
poppy cell cultures. Transformed cell lines expressing antisense
BBE or antisense CYP80B1 constructs and
displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed
reduced accumulation of benzophenanthridine alkaloids compared with
control cultures transformed with a 
-glucuronidase gene. Pathway
intermediates were not detected in any of the transformed cell lines.
The suppression of benzophenanthridine alkaloid biosynthesis using BBE
or CYP80B1 antisense RNA constructs also reduced the growth rate of the
cultures. Two-dimensional 1H-nuclear magnetic resonance and
in vivo 15N-nuclear magnetic resonance spectroscopy showed
no difference in the abundance of carbohydrate metabolites in the
various transgenic cell lines. However, transformed cells with reduced
benzophenanthridine alkaloid levels contained larger cellular pools of
several amino acids including alanine, leucine, phenylalanine,
threonine, and valine compared with controls. The relative abundance of
tyrosine, from which benzophenanthridine alkaloids are derived, was
less than 2-fold higher in antisense-suppressed cells relative to
controls. These results show that alterations in the metabolic flux
through benzophenanthridine alkaloid biosynthesis can affect the
regulation of amino acid pools. These data provide new insight into the
metabolic engineering of benzophenanthridine alkaloid pathways.
1
This work was supported by the Natural Sciences
and Engineering Research Council of Canada (grant to P.J.F.). S.U.P.
was the recipient of Bettina-Bahlsen Memorial, Graduate Faculty
Council, and J.B. Hyne Graduate Scholarships and a Dean's Special
Doctoral Scholarship offered through the University of Calgary.
2
Present address: Agriculture and Agri-Food Canada,
Saskatoon Research Centre, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2.
*
Corresponding author; e-mail pfacchin{at}ucalgary.ca; fax
403-289-9311.
© 2002 American Society of Plant Physiologists
