The Source Of Salicylic Acid-elicited H₂O₂and Its Effect On The Biosynthesis Of Phenolic Acids In Salvia Miltiorrhiza Bunge

The sources of H2O2in salicylic acid (SA)-induced biosynthesis of phenolic acids inSalvia miltiorrhiza were investigated. S. miltiorrhiza was treated with NADPH oxidaseinhibitors of diphenyleneiodonium (DPI) or imidazole (IMD), polyamine oxidase (PAO)inhibitors of quinacrine and guazatine, and peroxidase (POD) inhibitor of salicyl hydroximicacid (SHAM) or NaN3, and then subjected to SA treatment. The activities of oxidases and thecontent of H2O2were determined. The expression of phenylalanine ammonia-lyase (PAL)and the contents of caffeic acid (CA), rosmarinic acid (RA) and salvianolic acid B (Sal B)were analyzed to discuss the effect of H2O2derived from different sources on phenolic acidproduction.1．0.5mM SA could induce the generation of H2O2in leaves of S. miltiorrhiza. Theactivity of NADPH oxidase had also been improved. When applied with IMD and DPI, theactivity of NADPH oxidase was suppressed effectively, and the content of H2O2wasdecreased as the same level as control group, suggesting that NADPH oxidase was one of thesources of SA induced-H2O2.2. SA could increase the activity of ionic and covalent POD located in cell wall. Theactivity of ionic POD showed two peaks in2h and6h, respectively. The activity of covalentPOD had two peaks at2h and5h, respectively. The ionic POD was more sensitive after SAtreatment, the first peak of SA-induced POD activation was earlier than the second peak ofH2O2burst, and NaN3or SHAM inhibited the SA-induced H2O2production, suggesting thatPOD was involved in SA-induced H2O2.3. SA also increased the activity of PAO which use Spm and Spd as substrate, and theenzyme activity peaked at2hours. However, the activity of diamine oxidase which uses Putas substrate was not detected. Quinacrine inhibited the activity of PAO as well as the contentof H2O2, suggesting that SA-induced H2O2might be derived from PAO.4. H2O2generated by NADPH oxidase, POD, and PAO upregulated PAL expression. The effect of H2O2derived from NADPH oxidase showed the most significant effect. H2O2derived from three sources showed no significant inhibitory effect on CA production, whiledecreased the generation of RA and Sal B, indicating that H2O2generated by NADPH oxidase,POD, and PAO mediated the biosynthesis of RA and Sal B. IMD showed the most significantinhibitory effect, suggesting that NADPH oxidase was the main source of H2O2involved inphenolic acids biosynthesis among three oxidases.