Coumarins (1,2-benzopyrones) are ubiquitously found in higher plants where they originate from the phenylpropanoid pathway. They contribute essentially to the persistence of plants being involved in processes such as defense against phytopathogens, response to abiotic stresses, regulation of oxidative stress, and probably hormonal regulation. Despite their importance, major details of their biosynthesis are still largely unknown and many P450-dependent enzymatic steps have remained unresolved. Ortho-hydroxylation of hydroxycinnamic acids is a pivotal step that has received insufficient attention in the literature. This hypothetical P450 reaction is critical for the course for the biosynthesis of simple coumarin, umbelliferone and other hydroxylated coumarins in plants. Multiple P450 enzymes are also involved in furanocoumarin synthesis, a major class of phytoalexins derived from umbelliferone. Several of them have been characterized at the biochemical level but no monooxygenase gene of the furanocoumarin pathway has been identified yet. This review highlights the major steps of the coumarin pathway with emphasis on the cytochrome P450 enzymes involved. Recent progress and the outcomes of novel strategies developed to uncover coumarin-committed CYPs are discussed.

Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer's type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.

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.

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.