Effect Of Medium Calcium And Calcium Mobilization Triggered By Salicylic Acid On The Biosynthesis Of Rosmarinic Acid In Salvia Miltiorrhiza Suspension Cultures

In this paper, the effect of medium Ca²⁺and Ca²⁺mobilization triggered by salicylic acid on synthesisof intracellular RA and activities of related enzymes （PAL and TAT） in Salvia miltiorrhiza suspensioncultures was investigated. In addtion, the medium alkalinization in Salvia miltiorrhiza suspensioncultures treated with salicylic acid and the effect of Ca²⁺in this process were studyed, aiming atexpounding the biological function of Ca²⁺in secondary metabolism of Salvia miltiorrhiza suspensioncultures and providing theory evidence for artificial regulation of secondary metabolism.1. The synthesis of intracellular RA requiring medium Ca²⁺absolutely was significantly dependent onthe medium Ca²⁺concentration. In the test concentration range, at the optimal Ca²⁺concentration of10mmol/L, a maximal RA content was reached. However, The synthesis of RA was inhibited when mediumCa²⁺concentration was increased to50mmol/L. The variation of the activity of PAL and TAT, two keyenzymes of the two branch of RA, could be affected by the concentration of Ca²⁺in culture medium. Thechange of their activity occured prior to the accumulation of RA, which suggested both of the keyenzymes were involved in the synthesis of RA. Meanwhile, the enzymic action of PAL was more distinctthan TAT. The treatment of VP and A23187, respectively, indicated that thesynthesis of RA impacted by theconcentration of Ca²⁺in the culture medium results from the increase of intracellular Ca²⁺, and the flowof Ca²⁺from the extracell to the intracell could also participate in this process.2. LaCl3and VP or2-APB and LiCl, two different groups of calcium channel antagonist, maysignificantly inhibit the synthesis of RA induced by salicylic acid when they were used alone. However,The suppression effect from LaCl3or VP on synthesis of RA induced by salicylic acid was better thanthat from LiCl or2-APB. A23187and Ca²⁺feeding could promote the synthesis of RA. When two types ofcalcium channel inhibitor （LaCl3and2-APB） were used together, the synthesis of RA induced bysalicylic acid was more largely suppressed even if higher than that of blank control. In conclusion, thecalcium from both extracell and intracell calcium store participated in the synthesis of RA induced bysalicylic acid. In addition, the calcium from extracell plays a more dominant role in this process.3. Salicylic acid could induce significant medium alkalinization in Salvia miltiorrhiza culture. VP andLaCl3or LiCl and2-APB, two different groups of calcium channel antagonist, significantly inhibited themedium alkalinization induced by salicylic acid when they were used alone. However, the suppressioneffect of VP or LaCl3on medium alkalinization induced by salicylic acid was better than that of LiCl or2-APB. When two types of calcium channel inhibitor （LaCl3and2-APB） were used together, themedium alkalinization induced by salicylic acid was completely suppressed even reduced the pH in medium. On the other hand，A23187could promote the medium alkalinization. Based on the results above,we speculated that Salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture,dependenting on the calcium from both extracell and intracell. What’s more, the calcium from extracellplays a more dominant role in this process.