Abstract
BackgroundFuranocoumarins 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens
We describe the identification and in vitro/in vivo enzymatic characterization of a new gene encoding CYP98A22, a p-coumaroyl ester 3'-hydroxylase from R. graveolens, which constitutes the first CYP98 characterized from a furanocoumarin-producing plant
Identification of a cyp98a orthologous gene in R. graveolens To identify cyp98a genes present in furanocoumarinproducing plants, we used a PCR-based approach and the CODEHOP strategy described by Morant et al [24]
Summary
Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. The adaptation of plants to their environment and their survival under stressing conditions, e.g., pathogenic attacks, requires secondary metabolites, such as polyphenols These molecules are broadly distributed in the plant kingdom with more than 8,000 phenolic structures currently known, reactions through the inhibition of cytochrome P450 (P450) enzymatic activities [4,5]. They interact with nucleic acids through the photocycloaddition of pyrimidic bases [6]. It is essential to understand the biosynthetic pathway of furanocoumarins, and to determine how the production of these molecules could be enhanced
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