Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase.
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 x 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.
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Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
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Ellis
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Douglas
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D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018749:RNA, Plant / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology*; D050564:Trans-Cinnamate 4-Monooxygenase; D014197:Trees / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D018749:RNA, Plant; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase