Stimulation of the proline cycle and anthraquinone accumulation in Rubia tinctorum cell suspension cultures in the presence of glutamate and two proline analogs

Abstract

Anthraquinone biosynthesis in Rubia tinctorum L. involves different metabolic routes. Chorismic acid, the end-product of the shikimate pathway, becomes the branch point between primary and secondary metabolism. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP ? generated by proline reduction from glutamate could act as a cofactor of the first enzymes of the PPP. This pathway generates erythrose-4-phosphate, the substrate of the shikimate pathway. The aim of the present work was to study the effect of the addition of glutamate and two proline analogs, azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid (T4C), on the PPP, the proline cycle, and anthraquinone production in R. tinctorum cell suspension cultures. The addition of 5 mM of glutamate enhanced both anthraquinone (up to 30%) and total phenolic content (12%), which correlated well with proline accumulation. Only the addition of 200 lM of T4C resulted in an increase in anthraquinone production, which was accompanied by a rise in the proline content. Neither the addition of glutamate nor proline analogs resulted in the induction of PPP, so this route was not a limiting factor as a carbon donor to the shikimate pathway.

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs

“…This negative effect was greater with 200 lM T4C This treatment was therefore excluded from further analyses. These observations are in accordance with the toxic effects of T4C reported by others (Tarmizi and Marziah 2000), and with our own previous results (Perassolo et al 2011). …”

“…These results show that the treatment of M. citrifolia with proline analogs, especially with T4C, was a successful strategy to induce AQ accumulation. These findings correlate well with our previous results with this analog in Rubia tinctorum, since AQ content increased by 27% after 7 days of culture when cell suspension cultures of this species were treated with 200 lM of T4C (Perassolo et al 2011). No significant differences were observed in the accumulation of total phenolics between treatments at 6 days of culture (Fig.…”

“…This lack of difference was observed both at 6 and 10 days of culture, suggesting that this metabolic pathway is not a limiting step for secondary metabolite production. We previously found similar results in R. tinctorum cell suspension cultures subjected to treatments with the same analogs (Perassolo et al 2011). …”

“…The concentrations of A2C and T4C were chosen according to previous reports (Yang and Shetty 1998;Perassolo et al 2011). A2C did not adversely affect biomass production but T4C did (data not shown).…”

Label-Free Proteomic Analysis of Smoke-Drying and Shade-Drying Processes of Postharvest Rhubarb: A Comparative Study

“…In rhubarb, anthraquinone and phenolic acids act as the main active ingredients (Agarwal et al, 2001;Krafczyk et al, 2008). As reported previously, the shikimate pathway and the polyketide pathway are the main biosynthetic pathways leading to the production of anthraquinones in higher plants (Han et al, 2001;Perassolo et al, 2011). There are two main ways for the accumulation of anthraquinone substances in plants.…”