Reactive Oxygen Species (ROS) and anthraquinones production in chitosan-elicitated Rubia tinctorum cell suspension cultures

PERASSOLO, M; BUSTO, VD; QUEVEDO, CV; CARDILLO, AB; MARTÍNEZ, CA; CUADRADO, V; MERINI, LJ; GIULIETTI, AM; RODRÍGUEZ TALOU, JULIÁN

Dalian, China

Simposio; 13th International Biotechnology Symposium and Meeting; 2008

Anthraquinones (AQs) are secondary metabolites derived from isochorismic acid, ¦Á-ketoglutaric acid and isopentenyl diphosphate [1]. Their production in Rubia tinctorum can be induced by elicitors [2]. The response to elicitation triggers the production of reactive oxygen species (ROS) and cell death. AQs synthesis in R. tinctorum cell suspension cultures elicitated by chitosan involves stimulation of PLC and PI3K, changes in intracellular Ca2+ concentration and MAPK activation [3]. In defense responses, the pentose phosphate pathway (PPP) is also stimulated. This route produces NADPH, an antioxidant protection against ROS. The proline cycle induces PPP by the generation of NADP+, cofactor of glucose-6-phosphate dehydrogenase (G6PD), the first and limiting enzyme in PPP. The aim of this work was to analize the relationship between elicitation, oxidative burst and cell death. R. tinctorum suspension cultures were treated with chitosan (200 mg/L), chitosan plus Sodium Nitroprussiate (SNP), chitosan plus Lanthanum Chloride and chitosan plus Diphenylene Iodonium (DPI). Cell viability, G6PD activity, AQs, proline, and extracellular H2O2 content were determined after chitosan elicitation. AQs increased after 24 and 48 hours of chitosan elicitation. Cell death increased after 6 and 24 hours, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulation were found in chitosan and chitosan-DPI treatments. Cultures treated with chitosan and lanthanum chloride showed lower levels of AQs and cell death than chitosan alone. SNP prevented chitosan effects on cell cultures, since a decrease in both cell death and H2O2 levels were observed. It can be assumed that higher cell viability caused by SNP addition is a consequence of its effect on H2O2 production. Proline levels decreased after treatment with chitosan, as well as G6PD activity, showing that in this case, PPP may not be induced by elicitation. References [1] Han, Y., Van der Heijden, R. and Verpoorte, R. 2001. Biosynthesis of anthraquinones in cell cultures of the Rubiaceae. Plant Cell, Tissue and Organ Culture. 67: 201-220. [2] Vasconsuelo, A.A., Giuletti, A.M., G. Picotto, G., Rodr¨ªguez Talou J. and Boland, R. 2003. Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures. Plant Science. 165: 429¨C436. [3] Vasconsuelo, A. A. and Bolan, R. Molecular aspects of the early stages of elicitation of secondary metabolites in plants. 2007. Plant Science. 172: 861-875.