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

PERASSOLO M; QUEVEDO CV; BUSTO, V.; CARDILLO AB; MARTÍNEZ CA; CUADRADO VL; MERINI LJ; GIULIETTI A. M; RODRÍGUEZ TALOU J

Dalian, China

Simposio; 13th International Biotechnology Symposium and Meeting; 2008

International Biotechnology Symposium

Anthraquinones (AQs) are secondarymetabolites derived fromisochorismic acid, -ketoglutaric acid and isopentenyl diphosphate (Han et al., 2001). Their production in Rubia tinctorum can be-ketoglutaric acid and isopentenyl diphosphate (Han et al., 2001). Their production in Rubia tinctorum can beHan et al., 2001). Their production in Rubia tinctorum can be Abstracts / Journal of Biotechnology 136S (2008) S345–S355 S351S351 induced by elicitors (Vasconsuelo et al., 2003). 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 (Vasconsuelo and Bolan, 2007). 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 analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.Vasconsuelo et al., 2003). 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 (Vasconsuelo and Bolan, 2007). 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 analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.R. tinctorum cell suspension cultures elicitated by chitosan involves stimulation of PLC and PI3K, changes in intracellular Ca2+ concentration and MAPK activation (Vasconsuelo and Bolan, 2007). 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 analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.2+ concentration and MAPK activation (Vasconsuelo and Bolan, 2007). 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 analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.Vasconsuelo and Bolan, 2007). 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 analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.+, cofactor of glucose-6- phosphate dehydrogenase (G6PD), the first and limiting enzyme in PPP. The aim of this work was to analyze 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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.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 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.2O2 content were determined after chitosan elicitation. AQs increased after 24 and 48 h of chitosan elicitation. Cell death increased after 6 and 24 h, which correlated with enhanced H2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.2O2 production. The addition of DPI resulted in decreased chitosan-induced cell death and H2O2 production. Similar levels of AQs accumulationwere 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.2O2 production. Similar levels of AQs accumulationwere 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.2O2 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.2O2 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.