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Plant Physiol, May 2001, Vol. 126, pp. 317-329
Functional Characterization and Subcellular Localization of
Poplar (Populus trichocarpa × Populus
deltoides) Cinnamate 4-Hydroxylase1
Dae Kyun
Ro,
Nancy
Mah,
Brian E.
Ellis, and
Carl J.
Douglas*
Department of Botany (D.K.R., N.M., C.J.D.) and
Biotechnology Laboratory and Faculty of Agricultural Sciences (B.E.E.),
University of British Columbia, Vancouver, British Columbia, Canada V6T
1Z4
Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450
monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a
phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A
full-length cDNA encoding C4H was isolated from a hybrid poplar
(Populus trichocarpa × P.
deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was
most highly expressed in developing xylem. C4H
expression was also strongly induced by elicitor-treatment in poplar
cell cultures. To verify the catalytic activity of the putative C4H
cDNA, two constructs, C4H and C4H fused to the FLAG epitope
(C4H::FLAG), were expressed in yeast. Immunoblot
analysis showed that C4H was present in the microsomal fraction and
microsomal preparations from strains expressing both enzymes
efficiently converted cinnamic acid to p-coumaric acid
with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein
(GFP) gene was engineered and stably expressed in Arabidopsis. Confocal
laser microscopy analysis clearly showed that in Arabidopsis the
C4H::GFP chimeric enzyme was localized to the ER. When
expressed in yeast, the C4H::GFP fusion enzyme was also
active but displayed significantly lower specific activity than either
C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These
data definitively show that C4H is localized to the ER in planta.
1
This work was supported by the Natural Science
and Engineering Research Council of Canada (to C.J.D.) and by a
University Graduate Fellowship from the University of British Columbia
(to D.K.R.).
*
Corresponding author; e-mail cdouglas{at}interchange.ubc.ca; fax
604-822-6089.
© 2001 American Society of Plant Physiologists
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