Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine

Abstract

Isolated vacuoles from ajmalicine-producing cell suspensions of Catharanthus roseus accumulated the alkaloid ajmalicine. Dissipation of the transtonoplast pH gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. Addition of Mg-ATP resulted in a higher ajmalicine accumulation. Serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. Ajmalicine was converted in vitro into serpentine by horseradish basic peroxidases (EC 1.11.1.7). In cultured cells there was a striking conformity between the time course of serpentine content and that of the activity of basic peroxidases. Ajmalicine was converted efficiently into serpentine by basic peroxidases extracted from vacuoles and by intact isolated vacuoles. The results are consistent with the hypothesis that ajmalicine accumulates by an ion-trap mechanism and that the accumulated ajmalicine is converted into serpentine inside the vacuoles. By the transformation of ajmalicine into the charged serpentine a trap is created to retain the alkaloids more efficiently in the vacuole.

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst

“…In T. chinensis cells exposed to low-energy ultrasound, ROS was involved as an upstream event for activation of jasmonic acid synthesis and both events were partially implicated in taxol biosynthesis regulation (Wu and Ge, 2004). Particularly, ROS is related to indole alkaloid biosynthesis, since the oxidation of the monoterpenoid indole alkaloids as ajmalicine to serpentine is catalyzed by peroxidases involving H 2 O 2 (Blom et al, 1991). Our results suggest that in U. tomentosa cells a NAD(P)H oxidase and/or peroxidases could be implicated in H 2 O 2 production.…”

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

“…This research raised new questions into the specificity of these interactions due to the finding that inner-petal AVIs of lisianthus specifically bound a minor subset of the total anthocyanins (the 3,5-diglycosides) rather than the acylated triglycosides that are the major forms present in inner petal vacuoles. A contrasting mechanism of vacuolar trapping is that observed for ajmalicine, which is converted to a charged form, serpentine, in the vacuole [56]. Pyrrolizidine alkaloids are retained in the vacuole in an N-oxide form, while the non-oxide forms can be taken up by vacuoles but are rapidly lost [57].…”

Cloning and Characterization of a Candidate Gene from the Medicinal Plant Catharanthus roseus Through Transient Expression in Mesophyll Protoplasts

“…This gene codes for two alternative splicing transcripts/proteins, and given the high similarity of CroPrx3-a and CroPrx3-b with CroPrx1, it was hypothesized that they could be involved in TIA metabolism, since for instance the conversion of ajmalicine into serpentine has also been suggested to be mediated by an uncharacterized Prx (Blom et al, 1991;Sottomayor et al, 2004). Important for the implication of a Prx in TIA metabolism, is its subcellular localization in the vacuole, where the TIA substrates and products accumulate.…”