The Molecular Mechanisms In The Induction Of Soybean Flavones Biosynthesis And Characterization Of GmMYB100 Transcription Factor In Flavonoid Biosynthesis

Flavones,a major group of flavonoids in most plant tissues,play multiple roles in plant-environment interactions and human health.There are two flavone synthase genes,GmFNSII-1 and GmFNSII-2,encode the flavone synthase enzymes,which catalyze the conversion of flavanones into flavones.Although many genes involved in soybean flavones synthesis have been identified,the mechanism under the flavones inducation and regulation are not clear.In this study,the expression of the two soybean flavone synthase genes?GmFNSII-1and GmFNSII-2?and flavone aglycone content were significantly induced by methyl jasmonate?MeJA?,glucose,mannitol,and NaCl treatment,respectively.The GmFNSIIs promoter-GUS fusion vectors were constructed,and the results revealed that beta-glucuronidase activity in transgenic soybean hairy root were stimulated by MeJA,mannitol,sucrose and NaCl.The sequences of GmFNSIIs promoters were analyzed and characterized by promoter database?PLACE and PlantCARE?,and there are many stress-responsive cis-element in GmFNSII-1 and GmFNSII-2 promoter.In the GmFNSII-1promoter,a specific CGTCA-motif in the region?-979 bp to-806 bp?involved in the MeJA response was identified.Promoter deletion analysis of GmFNSII-2 revealed the presence of osmotic-responsive?-1143 bp to-767 bp?and glucose-repressive sequence elements?-767 bp to-475 bp?,which strongly supported the hypothesis that glucose induces soybean flavone production by acting as both an osmotic factor and a sugar signalling molecule simultaneously.Silencing of GmFNSIIs gene evidently reduced the production of flavone aglycones?apigenin,luteolin,and 7,4'-dihydroxyflavone?in hairy roots.The GmFNSII-RNAi roots that had a reduced level of flavones accompanied with more malondialdehyde and H₂O₂ accumulation were more sensitive to salt stress compared to those of the control,and it was concluded that flavones,as antioxidants,are associated with salt tolerance.Through the analysis and characterization of soybean transcription factors,123soybean flavone biosynthesis related transcription factors were identified.After the V1stage soybean seedlings were treated with 100?M MeJA for 24 h,and the expression levels of 123 transcription factors were detected by realtime PCR.The results revealed that the expression level of 63 genes were induced by MeJA and 14 genes was inhibited in soybean leaves,while the transcript level of 84 genes increased and 6 genes decreased in soybean roots.After the treatment of MeJA test,44 genes from 123 transcription factor were treated with salicylic acid?500?M for 12 h?,sucrose?0.4 M for 24 h?and Bradyrhizobium?USDA110 incubated with one week?.The expression level of 44 genes were analyzed,and 14 genes from 44 transcription factors were characterized.Finally the genomic DNA and cDNA of eight genes from 14 transcription factors were cloned successfully,and four ones?TF54,TF62,TF88 and TF100?from the eight genes exhibited transactivation activity in yeast experiment.TF100 gene,encode R2R3_MYB group protein,was named GmMYB100.The transcript level of GmMYB100 were high in soybean flower,leaves and early development stage embryo.The molecular weight of GmMYB100 protein is about 26.16 kDa,which mainly located in nucleus.There is a conserved amino acid motif?[D/E]LX2[R/K]X3LX6LX3R?in the R3 domain of GmMYB100,and the motif structure play role in the interaction of MYB protein and bHLH protein.A flavonoid-repress related amino acid motif is also identified in GmMYB100,and we speculated that GmMYB100 may negatively regulate soybean flavonoid biosynthesis.In GmMYB100 over-expression transgenic soybean hairy roots,the transcript level of GmCHS7,GmCHS8,GmCHI,GmIFS1 and GmF3H decreased significantly compared to the control ones,which lead to the decrease of soybean isoflavonoid content.However,the expression of GmCHS7,GmCHS8,GmCHI,GmFNSII-1 and GmF3H increased rapidly in GmMYB100-RNAi transgenic soybean hairy roots compared with the vector controls,which resulted in the siginaficant increase of soybean isoflavonoid and flavones.In the GmMYB100 over expression transgeninc Arabidopsis,the expression of AtCHS,AtCHI,AtF3'H,AtF3H,AtDFR2 and AtFLS were up-regulated compared to the control ones,and the decrease of flavonol in GmMYB100-overexpression Arabidopsis was tested.Tobacco transient experiment revealed that the GmMYB100 protein repress GmCHS8 and GmIFS1 promoters,while activate GmF3H,GmFNSII-1 and GmANS promoters.The results indicated that GmMYB100 negatively regulated the expression of the genes related with soybean flavone biosynthesis.In summary,the flavones biosynthesis were stimulated by MeJA,and JA responsive element?CGTCA-motif?was found in GmFNSII-1 promoter.Glucose induces soybean flavone production by acting as both an osmotic factor and a sugar signalling molecule simultaneously.The flavones metabolism was induced by NaCl,and the flavone content effected soybean salt resistance.We also found that the expression of GmMYB100 was inhibited by MeJA,and transcription factor GmMYB100 negatively regulated the transcriptive level of genes related with flavone biosynthesis.