Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase

• Published in: Plant physiology (Bethesda) Vol. 126; no. 1; pp. 317 - 329
• Main Authors: Dae Kyun Ro, Nancy Mah, Ellis, Brian E., Douglas, Carl J.
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01-05-2001; American Society of Plant Biologists
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; Artificial Gene Fusion; Base Sequence; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Biosynthesis; Catalysis; Cinnamates; Complementary DNA; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochromes; DNA Primers; DNA, Complementary; Economic plant physiology; Enzymes; Epidermal cells; Fluorescence; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins; Luminescent Proteins - genetics; Metabolism; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Nutrition. Photosynthesis. Respiration. Metabolism; Organelles; Plant cells; Plant physiology and development; Proteins; RNA, Plant - metabolism; Sequence Homology, Amino Acid; Subcellular Fractions - enzymology; Trans-Cinnamate 4-Monooxygenase; Trees - enzymology; Yeasts; Lignin; Phenylpropanoid pathway; Metabolic pathway; Biosynthesis; Specific activity; Arabidopsis thaliana; Characterization; Cruciferae; Dicotyledones; Angiospermae; Hardwood forest tree; Molecular cloning; Localization; Aminoacid sequence; Unspecific monooxygenase; Enzyme; trans-Cinnamate 4-monooxygenase; Gene expression; Biological activity; Salicaceae; Isolation; Spermatophyta; Oxidoreductases; Experimental plant; Endoplasmic reticulum
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.126.1.317

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.