Effects Of Ca²⁺ Induced By Salicylic Acid On The Biosynthesis Of Phenolic Acids Secondary Metabolites In Young Seedings Of Salvia M1ltiorrhiza Bunge

Salvia miltiorrhiza Bunge beyond to the medicinal plants of Lamiaceae Salvia.salvianolic acid B （Sal B） is the active ingredient and index components of S. miltiorrhiza,and rosmarinic acid （RA） is the direct precursor compound in Sal B synthesis pathway. In thisstudy, young seedlings of S. miltiorrhiza were used to select optimal concentration andsampling time of SA in the phenolic acids secondary metabolites synthesis pathway; then usethe confocal laser scanning microscopy （CLSM） to observe the change of fluorescenceintensity of Ca²⁺in guard cell induced by SA at the cellular level. Combined with thetreatment of pharmacological agents of calcium inhibitor/calmodulin antagonist, investigatedthe effect of SA and calcium inhibitor/calmodulin antagonist to the relative fluorescenceintensity of the calcium、contents of secondary metabolites and key enzymes in phenolic acidssecondary metabolite synthesis. The results are as follows:1. SA improved the contents of phenolic acids secondary metabolites significantly.0.5、2.0and4.0mmol/L SA were used to deal with the S. miltiorrhiza leaves, and we found that0.5-4.0mmol/L SA could promote the accumulation of phenolic acids （Sal B and RA）, as wellas the activities of related enzymes （PAL and TAT）. Especially the inducing effects of2.0mmol L-1SA, the enzymes activities reached a peak at12h after induction, while secondarymetabolites at24h. These results made a clear on the optimal concentration and samplingtime of SA in the phenolic acids secondary metabolites synthesis pathway.2. SA could induce the calcium burst in the guard cells of S.miltiorrhiza. After thetreatment of SA in8.68min, relative fluorescence intensity of Ca²⁺reaches to the maximumvalue （459.36）, lasting for2-3min, and gradually decreased to the relative fluorescenceintensity （404） which is the value of SA started processing. Extracellular calcium antagonist（VP and LaCl3）, intracellular calcium inhibitor （LiCl）, the intracellular calmodulin antagonist（TFP） could inhibit the burst of Ca²⁺. The results demonstrated that SA could induce thecalcium mobilization in the guard cells of S.miltiorrhiza.3. Ca²⁺elicited by SA regulated the biosynthesis of phenolic acids secondary metabolites in young seedlings of S. miltiorrhiz. Extracellular, intracellular calcium antagonist and/orcalmodulin antagonist could inhibit the biosynthesis of Sal B and RA （the content is less thanthe control group and the SA induction group）, and their related enzymes activity. The resultsindicated that there is a direct relationship between calcium mobilization induced by SA andphenolic acids secondary metabolite biosynthesis. The possible mechanism is that SA as aelicitor could bind to plasma membrane specific receptor, then open the intracellular andextracellular calcium channel to increase the intracellular calcium concentration. Calcium, asan intracellular second messenger signaling cascade, can activate the activity of key enzyme（PAL and TAT）, then makes the nucleus encoded protein to synthesize secondary metabolites.