Preliminary Study On Physiological And Molecular Mechanism Of Betulin Accumulation Induced By Putrescine In Betula Platyphylla Suspension Cells

In this paper, we used birch suspension cells as experimental material and treated it with different concentration of exogenous putrescine for different time to invest the effect of putrescine on betulin accumulation in Betula platyphylla suspension cells, then the treatment conditions were optimized. On this basis, to investigate the mechanism of betulin accumulation induced by Put, the defence mechanism and nitrogen metabolism of cells treated with exogenous putrescine, and the changes of accumulation of betulin and NO induced by putrescine, SNP and cPITO were analyzed, proteome of birch suspension cells treated with putrescine, SNP and cPITO for 12h were studied as well. The main results were as follows:1. To invest effects of different concentration of exogenous putrescine on dry weight and betulin content,0.1mmol/L-10mmol/L putrescine was added to birch suspension cells for 3-48h, cell dry and betulin content and related gene expression level were analyzed. The results suggested 0.lmmol/L treated for 48h had strongest role in promoting cell growth; the content of betulin was most enhanced after 1mmol/L putrescine induction for 12 h,.2. To clear the relationship between defense physiology and accumulation of betulin under putrescine treatment,0.1mmol/L-5mmol/L putrescine was added to birch suspension cells for 3-48h, enzyme activities of SOD, POD, CAT and PAL were detected as well as their gene expression level in birch suspension cells were detected. O.1mmol/L and lmmol/L putrescine promoted activities of cell defense enzymes and PAL, and lmmol/L putrescine-treated cells showed greater enzyme activities than control cells, however,5mmol/L putrescine decreased enzyme activities of defense enzymes and PAL. The result described above showed that putrescine regulated the gene expression level of enzymes, protein post-translational modification or regulated enzyme activities directly influenced defense enzymes and PAL activities, indicated oxidative burst and phenylalanine pathway were connected with putrescine-induced betulin accumulation.3. To invest relationship between NO synthesis, nitrogen metabolism and betulin accumulation when treated with 0.1mmol/L-5mmol/L putrescine for 0.5-48h. O.lmmol/L and 1mmol/L putrescine promoted NO accumulation through regulating NR and NOS activities, 5mmol/L putrescine also increased NO content, but inhibited accumulation of betulin, we preliminarily inferred that the induction of NO by putrescine through NR/NOS pathway could participate in the accumulation of betulin in suspension cells, or high concentration NO interfered with promotion of betulin induced by putrescine. In addition, 1mmol/L putrescine enhanced correlation between nitrogen metabolism and betulin accumulation, indicated nitrogen metabolism maybe participate in induction of putrescine on betulin.4. To invest whether NO mediated induction of putrescine on betulin, lmmol/L putrescine, lmmol/L SNP,150 μmol/L were added to birch suspension cells for 12h, NO content, NR and NOS activities and betulin content were analyzied. The fact putrescine and SNP could increase NO content and betulin accumulation in birch suspension cells had be proved, treatment with cPITO inhibited the putrescine-induced accumulation of betulin in some degree, but not completely. These results indicate that NO mediates putrescine-induced betulin accumulation in birch suspension cells, but there maybe other signal pathways response to putrescine participated in promotion of betulin accumulation. The gene expression level ofβ-AS、LUS and CAS were in accordance with betulin content, these findings suggested that putrescine may exert its effect on betulin accumulation via changes in gene expression level of key synthetase in betulin biosynthesis pathway.5. iTRAQ was used to identify proteome in cells treated with putrescine, SNP and cPITO, 4718 proteins were indentified, of which 4360 proteins matched to GO database,4466 proteins were found in COG database and 3691 protein were annotated to 126 metabolic pathways. A total of differential protein between two treatments were 667, the functional analysis showed that putrescine and SNP induced accumulation of synthesis, transcription and transportation proteins connected with triterpenoid, while cPITO inhibited the promotion induced by putrescine. These findings above suggested that the effect of putrescine on betulin accumulation partly own to NO derived from NR/NOS as well as JA/SA signal pathway, partly dependent other pathway such as 14-3-3 protein family mediated brassinolide signal pathway or calcium signal pathway, to influence secondary metabolic.