Salicylic acid-induced cytosolic acidification increases the accumulation of phenolic acids in Salvia miltiorrhiza cells

Salicylic acid (SA) is an elicitor widely used to promote the synthesis of secondary metabolites. In order to determine the relevance between SA-induced pH changes and secondary metabolite synthesis, Salvia miltiorrhiza suspension cells were exposed to either SA, fusicoccin (FC), sodium orthovanadate (OVA), methylamine (ME) and their combination (FC+SA and ME+SA) for investigating the effect of cytosolic pH change on phenolic acids production, such as caffeic acid (CA), rosmarinic acid (RA), and salvianolic acid B (Sal B). The variations of cytosolic pH were sensed by using the fluorescent probe BCECF-AM, and combination with laser scanning confocal microscopic technique. Meanwhile, the genes expression of phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), and rosmarinic acid synthase (RAS) were determined using real time PCR. The contents of CA, RA, and Sal B were measured by high performance liquid chromatography. The results showed that SA decreased the cytosolic pH by inhibiting the activity of plasma membrane H+-ATPase. OVA induced cytosolic acidification too, while both FC and ME inhibited the acidification induced by SA. SA also up-regulated the genes expression of TAT, PAL, and RAS, and as a result enhanced the accumulation of those phenolic acids.