Agrobacterium-mediated transformation of Ruta graveolens L.

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.

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.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.