CYP98A22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens
Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis.
In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn’t affect the synthesis of scopoletin.
The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.
- Dai J, Mumper RJ: Plant Phenolics: Extraction, Analysis and Their Antioxidant and Anticancer Properties. Molecules 2010,15(10):7313–7352. CrossRef
- Beier RC, Oertli EH: Psoralen and other linear furocoumarins as phytoalexins in celery. Phytochemistry 1983,22(11):2595–2597. CrossRef
- Schuler MA, Berenbaum MR: Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate. Proc Natl Acad Sci U S A 2003,100(Suppl 2):14593–14598.
- Koenigs LL, Trager WF: Mechanism-based inactivation of cytochrome P450 2B1 by 8-methoxypsoralen and several other furanocoumarins. Biochemistry 1998,37(38):13184–13193. CrossRef
- Gravot A, Larbat R, Hehn A, Lièvre K, Gontier E, Goergen JL, Bourgaud F: Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: Cloning and characterization of a C4H from a psoralen producing plant - Ruta graveolens - Exhibiting low sensitivity to psoralen inactivation. Arch Biochem Biophys 2004,422(1):71–80. CrossRef
- Dall'Acqua F, Vedaldi D, Recher M: The photoreaction between furocoumarins and various DNA with different base compositions. Photochem Photobiol 1978, 27:33–36. CrossRef
- Adisen E, Karaca F, Oztas M, Gurer MA: Efficacy of local psoralen ultraviolet A treatments in psoriasis, vitiligo and eczema. Clin Exp Dermatol 2008,33(3):344–345. CrossRef
- Pereira LE, Villinger F, Wulff H, Sankaranarayanan A, Raman G, Ansari AA: Pharmacokinetics, toxicity, and functional studies of the selective kv1.3 channel blocker 5-(4-Phenoxybutoxy)Psoralen in rhesus Macaques. Exp Biol Med 2007,232(10):1338–1354. CrossRef
- Plumas JL, Drillat P, Jacob MC, Richard MJ, Favrot MC: Extracorporeal photochemotherapy for treatment of clonal T cell proliferations. Bull Cancer 2003,90(8–9):763–770.
- Kawase M, Sakagami H, Motohashi N, Hauer H, Chatterjee SS, Spengler G, Vigyikanne AV, Molnar A, Molnar J: Coumarin derivatives with tumor-specific cytotoxicity and multidrug resistance reversal activity. In Vivo 2005,19(4):705–711.
- Larbat R, Kellner S, Specker S, Hehn A, Gontier E, Hans J, Bourgaud F, Matern U: Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis. J Biol Chem 2007,282(1):542–554. CrossRef
- Larbat R, Hehn A, Hans J, Schneider S, Jugdé H, Schneider B, Matern U, Bourgaud F: Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis. J Biol Chem 2009,284(8):4776–4785. CrossRef
- Hehmann M, Lukacin R, Ekiert H, Matern U: Furanocoumarin biosynthesis in Ammi majus L.: cloning of bergaptol O-methyltransferase. Eur J Biochem 2004,271(5):932–940. CrossRef
- Vialart G, Hehn A, Olry A, Ito K, Krieger C, Larbat R, Paris C, Shimizu B, Sugimoto Y, Mizutani M, et al.: A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl coA 2’-hydroxylase activity (C2’H): a missing step in the synthesis of umbelliferone in plants. Plant J 2012, 70:460–470. CrossRef
- Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D: CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem 2001,276(39):36566–36574. CrossRef
- Sullivan ML, Zarnowski R: Red clover coumarate 3'-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid. Planta 2010,231(2):319–328. CrossRef
- Schoch GA, Morant M, Abdulrazzak N, Asnaghi C, Goepfert S, Petersen M, Ullmann P, Werck-Reichhart D: The meta-hydroxylation step in the phenylpropanoid pathway: A new level of complexity in the pathway and its regulation. Environ Chem Lett 2006,4(3):127–136. CrossRef
- Morant M, Schoch GA, Ullmann P, Ertunç T, Little D, Olsen CE, Petersen M, Negrel J, Werck-Reichhart D: Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat. Plant Mol Biol 2007,63(1):1–19. CrossRef
- Eberle D, Ullmann P, Werck-Reichhart D, Petersen M: cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis. Plant Mol Biol 2009,69(3):239–253. CrossRef
- Matsuno M, Nagatsu A, Ogihara Y, Ellis BE, Mizukami H: CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4′-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis. FEBS Lett 2002,514(2–3):219–224. CrossRef
- Matsuno M, Compagnon V, Schoch GA, Schmitt M, Debayle D, Bassard JE, Pollet B, Hehn A, Heintz D, Ullmann P, et al.: Evolution of a novel phenolic pathway for pollen development. Science 2009,325(5948):1688–1692. CrossRef
- Basson AE, Dubery IA: Identification of a cytochrome P450 cDNA (CYP98A5) from Phaseolus vulgaris, inducible by 3,5-dichlorosalicylic acid and 2,6-dichloro isonicotinic acid. J Plant Physiol 2007,164(4):421–428. CrossRef
- Moglia A, Comino C, Portis E, Acquadro A, De Vos RCH, Beekwilder J, Lanteri S: Isolation and mapping of a C3?H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress. Plant Cell Rep 2009,28(6):963–974. CrossRef
- Morant M, Hehn A, Werck-Reichhart D: Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biol 2002, 2:7. CrossRef
- Voinnet O, Rivas S, Mestre P, Baulcombe D: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 2003,33(5):949–956. CrossRef
- Lièvre K, Hehn A, Thi LeMinh T, Gravot A, Thomasset B, Bourgaud F, Gontier E: Genetic transformation of the medicinal plant Ruta graveolens L. by an Agrobacterium tumefaciens-mediated method. Plant Sci 2005,168(4):883–888. CrossRef
- Zobel AM, Brown SA: Influence of low-intensity ultraviolet radiation on extrusion of furanocoumarins to the leaf surface. J Chem Ecol (Historical Archive) 1993,19(5):939–952. CrossRef
- Mahesh V, Million-Rousseau R, Ullmann P, Chabrillange N, Bustamante J, Mondolot L, Morant M, Noirot M, Hamon S, De Kochko A, et al.: Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: Evidence of a candidate for chlorogenic acid biosynthesis. Plant Mol Biol 2007,64(1–2):145–159. CrossRef
- Pompon D, Louerat B, Bronnie A, Urban P: Yeast expression of Animal and Plant P450s in optimized redox environments. Methods Enzymol 1996, 272:51–64. CrossRef
- Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D: Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem 1997,272(31):19176–19186. CrossRef
- Jiang H, Morgan JA: Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae. Biotechnol Bioeng 2004,85(2):130–137. CrossRef
- Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H, Werck-Reichhart D: Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems. Arch Biochem Biophys 2000,379(1):161–169. CrossRef
- Hehn A, Morant M, Werck-Reichhart D: Partial recoding of P450 and P450 reductase cDNAs for improved expression in yeast and plants. Methods Enzymol 2002, 357:343–351. CrossRef
- Su V, Hsu B-D: Transient Expression of the Cytochrome p450 CYP78A2 Enhances Anthocyanin Production in Flowers. Plant Mol Biol Rep 2010,28(2):302–308. CrossRef
- Su V, Hsu BD: Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers. Biotechnol Lett 2003,25(22):1933–1939. CrossRef
- Nafisi M, Goregaoker S, Botanga CJ, Glawischnig E, Olsen CE, Halkier BA, Glazebrook J: Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis. Plant Cell 2007,19(6):2039–2052. CrossRef
- Ro DK, Mah N, Ellis BE, Douglas CJ: Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 2001,126(1):317–329. CrossRef
- Wang W, Li S, Zhao X, Lin B, Du Y: Determination of six secondary metabolites including chlorogenic acid in tobacco using high performance liquid chromatography with coulometric array detection. Zhongguo Hua Xue Hui 2007,25(6):848–852.
- Nair RB, Xia Q, Kartha CJ, Kurylo E, Hirji RN, Datla R, Selvaraj G: Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast. Plant Physiol 2002,130(1):210–220. CrossRef
- Ogunkanmi AB, Tucker DJ, Mansfield TA: An improved bio-assay for abscisic acid and other antitranspirants. New Phytol 1973, 72:277–282. CrossRef
- Leiss KA, Maltese F, Choi YH, Verpoorte R, Klinkhamer PGL: Identification of Chlorogenic Acid as a Resistance Factor for Thrips in Chrysanthemum. Plant Physiol 2009,150(3):1567–1575. CrossRef
- Kai K, Mizutani M, Kawamura N, Yamamoto R, Tamai M, Yamaguchi H, Sakata K, Shimizu BI: Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J 2008,55(6):989–999. CrossRef
- Brown SA: Synthesis of daphnetin in Daphne mezerum. Z Naturforsch 1986,41(c):247–252.
- Brown SA, March RE, Rivett DEA, Thompson HJ: Intermediates in the formation of puberulin by Agathosma puberula. Phytochemistry 1988, 27:391–395. CrossRef
- Kai K, Shimizu B, Mizutani M, Watanabe K, Sakata K: Accumulation of coumarins in Arabidopsis thaliana. Phytochemistry 2006,67(4):379–386. CrossRef
- Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin H, Chiou TJ, Katagi H, Dewbre GR, et al.: Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 2000,22(6):531–541. CrossRef
- Tremblay R, Wang D, Jevnikar AM, Ma S: Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 2010,28(2):214–221. CrossRef
- Hoffmann L, Besseau S, Geoffroy P, Ritzenthaler C, Meyer D, Lapierre C, Pollet B, Legrand M: Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell 2004,16(6):1446–1465. CrossRef
- CYP98A22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens
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BMC Plant Biology
- Online Date
- August 2012
- Online ISSN
- BioMed Central
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- Author Affiliations
- 1. Université de Lorraine UMR 1121, Agronomie et Environnement Nancy-Colmar, ENSAIA 2 avenue de la forêt de Haye, 54505, Vandœuvre-lès-Nancy, France
- 2. INRA UMR 1121, Agronomie et Environnement Nancy-Colmar, ENSAIA 2 avenue de la forêt de Haye, 54505, Vandœuvre-lès-Nancy, France
- 3. CNRS UPR 2357 28 rue Goethe, Strasbourg, 67000, France