The Spatio-temporal Change Of H₂O₂and O₂Induced By Salicylic Acid In Salvia Miltiorrhiza Leaves

Hydrogen peroxide and superoxide anion radicals are two kinds of important reactiveoxygens in plants. They not only affect plants’ growth and development, but also regulatesecondary metabolism and defense responses as signal molecular in plants. This paper usedSalvia miltiorrhiza Bunge as the materials and salicylic acid (SA) as the exogenous inducer.Using titanous sulfate colorimetry detected the change of the content of H2O2and O2·-andusing DAB（3,3-diaminobenzidine）and NBT(nitroblue tetrazolium) methods located theaccumulation site of H2O2and O2·-. The paper also observated the accumulation anddistribution of H2O2in subcellular level by transmission electron microscope using ceriumchloride staining method. We getted the following conclusions:(1)Treatment of Salvia miltiorrhiza Bunge with SA resulted in an increase of the contentof H2O2.0.25、0.5、1.0and2.0mmol/L SA were used to deal with Salvia miltiorrhiza Bungeand we found that0.25~2.0mmol/L SA could all improve the content of H2O2. Especially theinducing effect of0.5mmol/L SA was the strongest and the content of H2O2reached a peak at4.0h after induction. NADPH oxidase inhibitor(DPI and IMD), peroxidase inhibitor(NaN3and salicylhydroxamic acid) and polyamine oxidase inhibitor(quinacrine) could inhibit thebrust of H2O2, but they could not inhibit it completely. These results demonstrated that SAimproved the content of H2O2through multiple channels.(2) Treatment of Salvia miltiorrhiza Bunge with SA resulted in an increase of the O2·-burst rate.0.25~2.0mmol/L SA could all improve the O2·-burst rate, especially the inducingeffect of0.5mmol/L SA was the strongest and the burst rate of O2·-reached a peak at2.0hafter induction.(3) It’s found that O2·-were mainly distributed in leaf main veins, lateral veins andmesophyll cells by using NBT method. With O2·-gushing quantity rising, its accumulationplaces extended gradually from the base of veins to the top of veins and mesophyll cells andthe blue spots became more intensive. H2O2are mainly distributed in leaf main veins andlateral veins by using DAB method.(4) It’s found that the accumulation of H2O2mainly included cell wall, cell membrane,vacuole membrane, intercellular space and chloroplast by transmission electron microscope using cerium chloride staining method. During the period of SA treatment, H2O2were locatedat cell membrane and vacuole membrane after inducing1.0h and the CeCl3sediment becamemore intensive in each of the leaf area after inducing4.0h.These above results revealed the accumulation and distribution of H2O2and O2·-fromcellular and subcellular level in Salvia miltiorrhiza Bunge leaves induced by SA. It’s afoundation for exploring the signal response and function mechanism of reactive oxygensmediated SA regulating the biosynthesis of secondary metabolism.