| Nitric oxide(NO)is an important signaling molecule that mediates the synthesis of secondary metabolites in plant cells.Recent studies have found that NO can regulate a variety of physiological processes in plants through protein S-nitrosylation,including plant stress,signal transduction and metabolic processes,et al.Previous studies in our laboratory found that protein S-nitrosylation-mediated the triterpenoid accumulation in suspension cells or seedlings of Betula platyphylla,but its target proteins mediated triterpenoid accumulation in Betula platyphylla are not clear.For this research,the effect of the diurnal rhythm,the denitrification way and the gene expression level of BpZFP9 on triterpenoid accumulation were analyzed.The main findings were as follows:Gene expression of lupeol synthase(LUS),one of key enzymes in triterpenoid biosynthesis,and zinc finger protein genes(ZFP1～10)increased in suspension cells and S-nitrosoglutathione reductase(GSNOR)transgenic birch plants by RNAi silencing under sodium nitroprusside(SNP,NO donor)treatment,and gene expression level of BpZFP7 and BpZFP9 were the highest in all being tested zinc finger protein genes.Thus,NO or RSNO significantly increased the gene expression level of BpZFP7,BpZFP9 and BpLUS genes.In three daily cycles,gene expression level of BpZFP9 and RSNO content reached the highest at 18:00 or 3:00 in birch leaves,respectively.Gene expression level of BpGSNOR,reduced glutathione(GSH)content and glutathione reductase(GR)activity peaked at 21:00.The diurnal variation of triterpenoid content showed a "ladder" pattern and maintained a high level from 18:00 to 6:00,and gene expression level of BpSS,BpLUS and BpCAS,key enzymes in triterpenoid biosynthesis,also peaked during 18:00-6:00.Thus,triterpenoid content was basically consistent with the gene expression of BpZFP9.The content of RSNO,gene expression of BpZFP9 and BpLUS in suspension cells treated by N6022 or ARF(inhibitors of key enzyme in denitrification pathway)were significantly increased than that of control,but gene expression of BpGSNOR decreased.It inferred that protein S-nitrosylation of ZFP9 mediated the synthesis of triterpenoids in Betula platyphyllaGene expression of BpZFP9 and BpLUS had the same trend in birch leaves infected by agrobacterium with overexpression or silencing vector of BpZFP9.220 stable birch transgenic lines with overexpression of BpZFP9 were obtained,17 positive lines were verified by PCR.Among of them,gene expression of BpZFP9 increased than that of wild birch plant.In conclusion,protein S-nitrosation of BpZFP9 mediated the synthesis of triterpenoids in Betula platyphylla. |
