Hydrogen Oxide And Nitric Oxide And Involoved In The Signal Transduction Of Salicylic Acid-induced Salvianolic Acid B Biosynthesis In Salvia Miltiorrhiza Cell Cultures

Salvia miltiorrhiza Bunge is a commonly used traditional Chinese medicine for improving body function, such as improving blood flow and promoting circulation, as well as for the treatment of myocardial infarction, angina pectoris and other cardiac symptoms. Salvianolic acid B (Sal B), a main phonetic aicd in S. miltiorrhiza Bunge aroused scientist’s interest in the last twenty years because of their notable pharmacological activities and the transditional use of herbs by decocting with water.The biotechnological technology production of valuable secondary metabolites by elicitor treatment is an effective way to extraction of whole plant material, which is more economically feasible way and environmental friendly. S. miltiorrhiza callus cultures provide a efficient and stable platform for effective constituent accumulations by using elicitation with suitable abiotic and biotic elicitors. In this study, we use salicylic acid B as elicitor to study the effects of different concentrations SA and different treatment time on the synthesis of Sal B in S. miltiorrhiza suspension cultured cells, and get the following conclusions:1. Treatment of S. miltiorrhiza cultured cells with SA resulted in an increase of the activity of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) and accumulation of Sal B. Treatment suspension cultured callus with22mg/L SA in the eighth day for two days can effectively promote the accumulation of Sal B in S. miltiorrhiza.2. H2O2acts as a signal molecule is involved in the signal transduction of SA-induced Sal B biosyhthesis in S. miltiorrhiza cell cultures. Treatment of S. miltiorrhiza cultured cells with S A resulted in an increase of H2O2content, the increase of the activity of PAL and TAT and accumulation of Sal B. Exogenous application of10-30mmol/L H2O2was found to effectively increase PAL and TAT activity as well as the Sal B content. Catalase (CAT), a H2O2scavenger, eliminated the Sal B-accumulating effects of exogenous H2O2and SA. These indicated that H2O2may function as an upstream signaling molecule in the SA-induced accumulation of Sal B signal transduction pathway. Treatment with2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (DMTU), a chemical trap for H2O2, was observed to be effective in inhibiting SA-induced accumulation of Sal B. Imidazole (IMD) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H2O2formation in plant cells. IMD treatment strongly inhibited the accumulation of Sal B in cultured cells of S. miltiorrhiza, but the inhibitory effects of IMD can be partially reversed by the exogenous SA. This indicated that the accumulation of Sal B was blocked once the generation of H2O2by NADPH oxidase was inhibited, and H2O2mediated the SA-induced Sal B accumulation.3. NO acts as a signal molecule is involved in the signal transduction of SA-induced Sal B biosynthesis in S.miltiorrhiza cell cultures. Treatment S. miltiorrhiza cell culture with SA resulted in the increase of NO content, the increase of the activity of PAL and accumulation of Sal B. Exogenous application of0.1～0.5mmol/L SNP, a NO donor, was found to effectively increase PAL activity and the Sal B content. Treatment with L-α-aminooxy-β-phenylpropionic acid (L-AOPP), a specific PAL inhibitior, effectively inhibited the increase of PAL activity and the accumulation Sal B, but do not inhibit the release of NO. These indicated that NO may function as up-stream signaling molecule in the SA-induced accumulation of Sal B signaling transduction pathway. Treatment with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO), a specific NO scavenger, was observed to be effective in inhibiting SA-induced accumulation of Sal B. Nω-nitro-L-arginine (L-NNA) strongly inhibits the activity of NOS enzyme inhitors, which is one of the main sources of NO formation in plant cells. L-NNA treatment strongly inhibited the accumulation of Sal B in cultured cells of S. miltiorrhiza, but the inhibitory effects of L-NNA can be partially reversed by the exogenous SA. This indicated that the accumulation of Sal B was blocked once the generation of NO through NOS was inhibited, and NO mediated the SA-induced Sal B accumulation.4. H2O2and NO act as signal molecule are involved in the signal transduction of SA-induced Sal B biosynthesis in S. miltiorrhiza cell cultures. In this study we used SNP、H2O2、DMTU、IMD、cPITO and L-NNA to treatment the S. miltiorrhiza cultured cells, the rusults showed that in the process of SA-induced the accumulation of Sal B can through the H2O2and NO-dependent or independent signaling pathways, as well as can by indendent H2O2and NO signaling pathways to mediate the accumulation of Sal B.