Influence Of Cytoplasmic Acidification On Salicylic Acid-induced Accumulation Of Phenolic Acids In Salvia Miltiorrhiza Suspension Cultures

In this study, the effect of Salicylic acid(SA)-induced cytoplasmic acidification in cultured S. miltiorrhiza cells was investigated. SA was added as elicitors to induce cytoplasmic acidification and H2O2 burst in S. miltiorrhiza cells. In order to investigate the effect of plasma membrane H+-ATPase(PM H+-ATPase) in SA-induced cytoplasmic acidification, the cultured S. miltiorrhiza cells were treated with PM H+-ATPase inhibitor(OVA) and PM H+-ATPase activator(FC) to change the activity of PM H+-ATPase. The cells were treated with NADPH oxidase inhibitor(IMD), H2O2 quenching agent(DMTU) to reduce the production of H2O2, in order to inspect the influence of SA-induced H2O2 on the process of cytoplasmic acidification. Then the effects of SA-induced cytoplasmic acidification on TAT, PAL and RAS expression, plus CA, RA and Sal B biosynthesis were detected respectively using q PCR and HPLC. The goal of this study was to elucidate the reason of how can SA induced cytoplasmic acidification, the relationship between H2O2 burst and cytoplasmic acidification induced by SA and the role of cytoplasmic acidification in secondary metabolites synthesis signaling pathways of phenolic acids. This test provides a theoretical basis for elucidating the signal transduction pathways of phenolic acid biosynthesis.Achieved the following main conclusions:1. SA treatment could induce cytoplasmic acidification in S. miltiorrhiza culture cells, and inhibit the activity of PM H+-ATPase; OVA inhibited the activity of PM H+-ATPase and caused cytoplasmic acidification; FC enhanced the activity of PM H+-ATPase and inhibited the cytoplasmic acidification induced by SA. These results suggested that one of the reason of cytoplasmic acidification was the inhibition of PM H+-ATPase by SA. SA-induced cytoplasmic acidification was accompanied with the medium alkalinization.2. SA treatment could induce significant H2O2 burst in S. miltiorrhiza culture cells. Exogenous H2O2 caused cytoplasmic acidification. DMTU and IMD inhibited the process of SA-induced cytoplasmic acidification, which shown that H2O2 mediated SA-induced cytoplasmic acidification; OVA treatment led the H2O2 to burst ahead of time. FC treatment inhibited the accumulation of H2O2 induced by SA, which suggested that SA-induced cytoplasmic acidification accelerate H2O2 burst. That was to say H2O2 level correlative with cytoplasmic acidification and they can function together to respond to SA stimulation.3. SA treatment increased the expression of TAT, PAL and RAS and the accumulation of caffeic acid(CA), rosmarinic acid(RA) and salvianolic acid B(Sal B). OVA caused cytoplasmic acidification with the same effects as SA on gene expression and accumulation of phenolic acids. FC and methylamine(ME) prevented SA-induced cytoplasmic acidification and prevented the promoter effects of SA on expression of related enzyme genes and accumulation of phenolic acids. These results suggest that SA causes cytoplasmic acidification by inhibiting PM H+-ATPase activity, therefore affecting TAT, PAL and RAS expression, which in turn mediate the accumulation of CA, RA and Sal B.In conclusion, SA triggers cytoplasmic acidification by inhibiting PM H+-ATPase activity. cytoplasmic acidification and H2O2 function together as signals, mediating the upregulation of gene expression of PAL,TAT,RAS, thus the accumulation of phenolic acids—CA、RA and Sal B as downstream secondary metabolites. This study provides a theoretical basis for elucidating the signal transduction pathways of phenolic acid biosynthesis.