We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts' vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1x10(6) cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.
Aniline Compounds ; chemistry ; Cell Culture Techniques ; Cellulase ; chemistry ; Fluorescent Dyes ; chemistry ; Protoplasts ; chemistry ; Salvia miltiorrhiza ; growth & development ; Xanthenes ; chemistry

In order to study the effects of Ca2+ in the biosynthesis of salvianolic acid B (Sal B) induced by salicylic acid (SA) in the young seedlings of Salvia miltiorrhiza, we used confocal laser scanning microscopy and high performance liquid chromatography to measure the change of relative fluorescence intensity of Ca2+ and the contents of Sal B induced by SA before and after the application of extracellular calcium channel inhibitors (VP and LaCl3), intracellular calcium channel inhibitor (LiCl), as well as intracellular calmodulin antagonist (TFP). SA could induce the calcium burst, and the Ca2+ peak could last to 2-3 min in the guard cells of S. miltiorrhiza, which prompted the biosynthesis of Sal B after the Ca2+ burst. Both Vp or LaCl3, and LiCl or TFP could inhibit the burst of Ca2+ and the biosynthesis of Sal B. The above results demonstrated that Ca2+ from the extracellular and the intracellular calcium store regulate the biosynthesis of Sal B elicited by salicylic acid in S. miltiorrhiz young seedlings.
Benzofurans ; metabolism ; Calcium ; metabolism ; Plant Leaves ; metabolism ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Seedlings ; metabolism ; Signal Transduction

Hydrogen peroxide (H2O2), one of reactive oxygen species, is widely generated in many biological systems, and it mediates various physiological and biochemical process in plants. To investigate the role of H2O2 as a signaling molecule in the process of salicylic acid (SA)-induced Salvianolic acid B (Sal B) accumulation, we separately inspected the cultured cells of Salvia miltiorrhiza with SA, H2O2, catalase (CAT), 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (DMTU) and Imidazole (IMD) to investigate the influence on the activity of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) and the accumulation of Sal B. Treatment of S. miltiorrhiza cells with SA resulted in an increase of H2O2, the increase of PAL and TAT and accumulation of Sal B. Exogenous application of 10-30 mmol/L H2O2 was found to effectively increase PAL and TAT activity as well as the Sal B content. CAT, a H2O2 scavenger, eliminated the Sal B-accumulating effects of exogenous H2O2 and SA. These indicated that H2O2 may serve as an upstream signaling molecule in the SA-induced accumulation of Sal B signal transduction pathway. Disposed by DMTU, a chemical trap for H2O2, as observed to be effective in inhibiting SA-induced accumulation of Sal B. IMD strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H2O2 formation in plant cells. IMD treatment strongly inhibited the accumulation of Sal B in cultured cells of S. miltiorrhiza, but the effects of IMD, can be partially reversed by the exogenous SA. The accumulation of Sal B was blocked once the generation of H2O2 by NADPH oxidase was inhibited, and H2O2 served as signaling molecule mediated the SA-induced Sal B accumulation.
Benzofurans ; metabolism ; Cell Culture Techniques ; methods ; Cells, Cultured ; Hydrogen Peroxide ; pharmacology ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; cytology ; metabolism ; Signal Transduction ; drug effects

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.
Cell Culture Techniques ; methods ; Cinnamates ; metabolism ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Cells ; metabolism ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; cytology ; growth & development ; metabolism ; Suspensions ; Tyrosine Transaminase ; metabolism

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.
Calcium ; pharmacology ; Cinnamates ; metabolism ; Culture Media ; Culture Techniques ; methods ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Salvia miltiorrhiza ; chemistry ; enzymology ; growth & development ; Tyrosine Transaminase ; metabolism

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.
Calcimycin ; pharmacology ; Calcium ; chemistry ; Calcium Channel Blockers ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Culture Techniques ; Culture Media ; chemistry ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Verapamil ; pharmacology