title: 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.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Université de Lorraine UMR 1121, Agronomie et Environnement Nancy-Colmar, ENSAIA 2 Avenue de la Forêt de Haye, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Fazeelat
authors.initials: F
authors.lastname: Karamat
authors.forename: Alexandre
authors.initials: A
authors.lastname: Olry
authors.forename: Sébastien
authors.initials: S
authors.lastname: Doerper
authors.forename: Guilhem
authors.initials: G
authors.lastname: Vialart
authors.forename: Pascaline
authors.initials: P
authors.lastname: Ullmann
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bourgaud
authors.forename: Alain
authors.initials: A
authors.lastname: Hehn
mesh_terms: D000595:Amino Acid Sequence; D002726:Chlorogenic Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D011564:Furocoumarins / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005822:Genetic Vectors; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D032105:Ruta / Q000201:enzymology / Q000235:genetics*; D012603:Scopoletin / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D011564:Furocoumarins; D002726:Chlorogenic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D012603:Scopoletin