Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.

Development of a novel system for producing ajmalicine and serpentine using direct culture of leaves in Catharanthus roseus intact plant.

Due to problems of production instability, the production of plant secondary metabolites using dedifferentiated cells (callus) is not always feasible on an industrial scale. To propose a new methodology, which does not use dedifferentiated cells, a novel system for producing useful secondary metabolites using the direct culture of intact plant leaves was developed. Catharanthus roseus was used as a model medicinal plant to produce terpenoid indole alkaloids (TIAs) by suspension culture of the leaves in the phytohormone-free MS liquid medium. Adjustment of the osmotic pressure (993 kPa at 25 degrees C) in the medium, light irradiation (60 micromol m(-2) s(-1)) and addition of glucose (10 g/l) were effective to promote the production of TIAs such as ajmalicine (Aj) and serpentine (Sp). On the basis of semi-quantitative RT-PCR analyses, it was revealed that the culture conditions promoted gene expression of enzymes in the TIA pathway in the cultured leaves. By feeding glucose (10 g/l) on day 10 of the culture period, Aj was produced at a concentration of about 18 mg/l and Sp was produced at a concentration about 11-fold that of the control. These results represent the first step in the development of a novel production system for plant secondary metabolites.