| In this study,anther suspension cells of Betula platyphylla were selected as materials.The optimal concentration of sodium nitroprusside(SNP,NO donor)for the anther cells to regulate biosynthesis of triterpenoid was filtered out and used in the follow-up experiment.The dynamic changes of triterpenoid content,cell viability,malondialdehyde(MDA),proline and antioxidant enzyme sysetem(including catalase/CAT,ascorbate peroxidase/APX and peroxidase/POD)were detected.To interpret the cross-talk of nitric oxide(NO)and reactive oxygen species(ROS)in regulating triterpenoid biosynthesis,contents of hydrogen peroxide(H2O2)and superoxide anion radical(O2·-)were detected to identify the changes of ROS level,and triterpenoid contents of anther suspension cells which were pretreated with diethylenbenzene(DPI,NADPH-oxidase inhibitor),sodium diethyldithiocarbamatre(DDTC,superoxide dismutase inhibitor)and catalase(CAT,scavenger)30 minutes earlier before SNP were added,respectively,were detected.In addition,analysis of triterpenoid contents of anther suspension cells under pretreatment of Ca2+ channel blocker and Ca2+ chelator were taken to reveal the function of Ca2+ signal pathway in exogenous NO regulated triterpenoid biosynthesis.Meanwhile,apoptosis of anther cells was also detected during treatment with exogenous NO to reveal relationships between secondary metabolite and apoptosis.The results are as follows:1 The effect of exogenous NO on triterpenoid concents,cell growth,activities of antioxidase and related gene experission levels in birch cell.0.0 mM,0.1 mM,1 mM,10 mM and 20 mM SNP promoted the contents of triterpenoid in birch while inhibiting the increase of fresh weight in the first 48 houes after treatment.The triterpenoid contents were 19.43%,40.14%,43.20%,30.89%and 12.05%higher than control by turns at 48 hours after treatment and fresh weight of cells were 28.90%,42.68%,45.42%,71.96%and 64.90%lower than control.Total triterpenoid content presented does-dependent effect with the concentration of SNP when the concentration of SNP bellowed 1 mM.1 mM was selected as the final concentration.The content of oleanolic acid in cells treated with 1 mM SNP was 5.69 folds to control at the 12th hour.The technology of promoting triterpenoid synthesis in Betula platyphylla by NO is as follow:1 mM SNP is added to liquid medium at the 8th day of 10-day cultivate cycle,the oleanolic acid content will reach the higheast level at the 12th hour after treatment while the total triterpenoid at the 48th hour.Exogenous NO could activate the defense system of birch within 0?6h,activities of CAT and PAL were 73.03%and 35.19%higher than control while the average activities of APX and POD were activated after 12 hours after treatment,which were 106.94%and 43.36%higher than control.Average contents of MDA and proline during Oh to 168h after treatment were 18.67%and 38.34%higher than control,indicating that exogenous NO caused a certain stress to anther cells of birch.Exogenous NO could affect triterpenoid content by regulating activities of key enzymes in triterpenoid biosyhthesis pathway in birch cells.The highest experission levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene(BpHMGR),deoxy-xylulose phosphate isomerase gene(BpDXR),squalene epoxidase gene(BpSQS),?-amyrenol gene(BpBPY)and cytochrome P450 enzyme gene(BpCYP716A)could reach 1.89-fold,11.31-fold,4.23fold,948.23-fold and 10.48-fold to control,at the 48th hour,24th hour,24th hour,6th hour and 6th hour after treatment respectively.Meanwhile,the experission level of calmodulin gene(BpCALM)reached 53.73-fold to control at the 6th hour after treatment,the experission level of nitrate reductase gene(BpNIA)reached 29.65-fold to control at the 24th hour,and the experission level of nitricoxide synthase gene(BpNOS)reached 2.10-fold to control at the 12th hour after treatment.Furthermore,exogenous NO showed up-regulated functions to antioxidase genes such as Cu-Zn-SOD(BpSODC,3.39 folds)and heme oxygenase gene(BpHO,4.43 folds).2 The effect of ROS on triterpenoid content induced by exogenous NOThe content of ROS increased after exogenous NO induced in suspension cells of birch.The content of H2O2 was 3.03-fold to control at the 6th hour after treatment.The superoxide anion radical(O2·-)content was 1.64-fold to control at the 12th hour after treatment.The oleanolic acid contents of cells at the 48th hour after pretreated with DPI and cells at the 12th hour after pretreated with CAT were 38.70%and 46.68%lower than that treated with SNP alone,while the oleanolic acid content of cells at the 12th hour after pretreated with DDTC was 38.87%higher than that treated with SNP alone.The data mentioned above showed that ROS mediate the process.Secondary metabolite synthesis of birch was induced by exogenous NO.ROS participated in the process which exogenous NO mediates genes expression in birch anther cell.The expression level of BpDXR gene reduced 46.41%when H2O2 was eliminated,otherwise,the one of BpDXR was increased by 322.81%for the accumulation of O2·-.The expression level of BpCALM was reduced by 63.14%because of the elimination of H2O2.The level of BpNIA expression was reduced by 74.65%after the partly inhibition of the resource of ROS.The results listed above demonstrated that NO induced the process of endogenous signal(Ca2+ and NO)synthesis.3 The effect of Ca2+ on triterpenoid accumulation induced by exogenous NOExogenous NO induced the content of Ca2+ to increase in Betula platyphylla at the 6th hour after treatment.The oleanolic acid contents of cells pretreated with nifedipine and LiCl were 68.84%and 65.53%lower than that of cells treated with SNP alone at the 12th hour after treatment,indicating that both intracellular calcium store and extracellular calcium store participated in the process that exogenous NO mediated the triterpenoid synthesis and inhibited the cell growth.The viability of cells pretreated with nifedipine was 41.41%lower than SNP group at the 12th hour after treatment,but differences of cell viability between cells treated with LiC and those with SNP were not significant,indicating that the downstream reactions of Ca2+signal pathway inhibited the reduction of cell activity caused by NO,especially extracellular calcium store.The blocking of Ca2+ signal pathway disturbed the initiation of antioxidant system.The activities of CAT,APX and POD were reduced by 76.46%,17.96%and 71.00%at most.The expression level of Mn-SOD gene(BpSODM)was reduced by 53.99%.At the same time,the content of the intracellular ROS was at most increased by 158.62%.The dates showed above indicated that Ca2+ signal pathway had ability to mediate the level of intracellular ROS through changing the activity of antioxidase and the expression levels of genes.The up-regulation effect of the key genes to synthesize triterpenoid induced by exogenous NO was inhibited when Ca2+ signal pathway were blocked.The expression level of BpBPY was reduced by 59.67%in nifedipine group and the expression of BpHMGR was reduced by 54.94%in BAPTA/AM group.The results suggested that Ca2+ could mediate the triterpenoid synthesis by controlling the expressions and activities of the key enzymes.The expression level of BpDXR was up-regulated by 108.49%in cells pretreated with LiCl,otherwise,the expression level of BpHMGR was reduced by 49.30%.From what has been discussed above,Ca2+ signal pathway regulates the gene expression by various patterns.4 The effect of exogenous NO on apoptosisExogenous NO not only regulated to synthesize triterpenoid,but also induced apoptosis in Betula platyphylla and increased the cell death rate.The rate of the cell death was 29.99%at the 48th hour after treatment,and the rate of apoptosis was 3.07 times as many as control.Induced by NO after 12 hours and 48 hours,the cell death rates reached the maximum which were 3.41 and 2.62 times as many as control at the 12th hour and the 48th hour after treatment,respectively.The changing trend of apoptosis was in accordance to that of oleanolic acid content.ROS and Ca2+ both participated in the course that exogenous NO induced the apoptosis and the cell death in Betula platyphylla.H2O2 was the main ingredient to regulate the apoptosis.O2·-played a key role in inducing cell death,and Ca2+ indirectly regulated the apoptosis. |
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