The Role Of H₂O₂in The Salicylic Acid Induced Antioxidant System In Salvia Miltiorrhiza Suspension Cultures

We studied the effects of the Salicylic acid (SA)-induced H2O2burst in Salviamiltiorrhiza cells, the inhibitors of chloroplast photosynthetic electron transport chain,NADPH oxidase and POD were used to block H2O2generation, the activity of H2O2on theactivities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were studied.The purpose of this study was to clear the effect of SA-induced H2O2, generated at thespecific sites, on the anti-oxidant system. This study will contribute to basic study of stressrespond mechanism of Salvia cultured cells, For the provision of basic research reveals SAstimulation caused by the reaction mechanism of Salvia miltiorrhiza suspension cultures.Achieved the following main conclusions:1. Salvia miltiorrhiza suspension cultures contained completed chloroplasts. In lightintensity (2000-3000Lx), callus grown loose green, chloroplast structure and function ofthe cell integrity. Under the transmission electron microscope, the chloroplast number rich,neatly arranged in the cell edge, grana lamellae closely arranged, clearly visible. Measuredby physiology indicators we found the content of chlorophyll a and chlorophyll b in Salviamiltiorrhiza suspension cultures ratio close to3:1, according with plant physiologycharacteristics.2. SA treatment could cause Salvia callus H2O2burst, and the optimal concentrationwas induced type characteristics.15.625-22.5mg·L-1SA could promote H2O2generationin Salvia miltiorrhiza suspension cultures generation, including20.83-22.5mg·L-1, highconcentration (25mg·L-1) of SA after induction could cause cell damage and lead toreduced synthesis. SA concentration of22.5mg·L-1could induce the strongest effect (2.3times the control group).3. SA treatment can stimulate the chloroplast produce H2O2, activate antioxidantsystem of Salvia miltiorrhiza suspension cultures. SA treatment increased antioxidantenzymes (SOD, CAT and POD) activities, membrane lipid peroxidation malondialdehydeand osmotic adjustment substance content, it also increased glutathione levels, with thediuron (DCMU) treatment, blocking chloroplast photosynthetic electron transport chain which to produce H2O2pathway inhibitor, three kinds of enzyme activities decreased,malondialdehyde and glutathione levels decreased. Polyacrylamide gel electrophoresisanalysis of isozymes result was consistent with the physiological test of three enzymes. SAcould induce chloroplast produce H2O2, activated antioxidant protection systems of Salviamiltiorrhiza suspension cultures to remove excess ROS, to ensure the normal metabolismof cells.4. SA treatment could induce NADPH oxidase and POD of Salvia miltiorrhizasuspension cultures, and produce H2O2, further cause the change of SOD, CAT and PODactivity, polyacrylamide gel electrophoresis analysis of isozyme bands consistent withphysiological test results; SA treatment increased malondialdehyde (MDA) andglutathione contents. After using the imidazole (the inhibitors of NADPH oxidase)andsodium azide (the inhibitors of POD) in Salvia miltiorrhiza suspension cultures treatment,the SA induced H2O2generation was blocked, and followed by the promoted activites ofSOD, POD and CAT, as well as the decreased levels of malondialdehyde and glutathione.SA induced H2O2produced from NADPH oxidase and POD in Salvia callus activatedantioxidant systems to remove excess ROS, maintain normal physiological activities.These results clearly revealed the impact of SA-induced H2O2produced specific sites onSalvia miltiorrhiza suspension cultures antioxidant system, the purpose is to provides atheoretical basis, which reveals the elicitor-induced reactions in Salvia miltiorrhizasuspension cultures resilience.