| Flavonoid biosynthesis is a complex process that is tightly regulated at transcription and posttranscription levels.Many transcription factors have been identified to play essential roles in regulating the biosynthesis of flavonoids.MYB-b HLH-WD40-repeat(MBW)transcriptional complexes are involved in the regulation of biosynthesis of anthocyanins,proanthocyanidins(PAs),and other biological processes.Among various types of MBW ternary complexes formed by different MYB,b HLH,and WD40-repeat transcription factors,MYB transcription factors are regarded as the core of regulatory network,which determine the specificity of the ternary complexes.Many MYB transcription factors had been identified,and the regulatory mechanisms wers also revealed in Arabidopsis.However,limited number of transcription factors has been characterized in leguminous plants,and the regulatory mechanism for them is just emerging.MtPAR,as a MYB type transcription factor,was characterized as a specific regulator for PA biosynthesis,as supported by its loss-of-function mutant seed coats displaying PA deficiency phenotype.However,the exact mechanism by which MtPAR regulates PA biosynthesis remain elusive,which became a serious drawback in MtPAR applications in metabolic engineering in breeding of bloat-free alfalfa varieties.On the other hand,research on flavonoid biosynthesis in soybean is greatly slowing due to the lack of soybean mutant libraries,although soybean seeds are well-known for its rich in isoflavones,anthocyanins,and PAs.So far,few systematic studies on soybean flavonoid biosynthesis and regulatory mechanism have been reported.Therefore,this research was mainly focused on the following two aspects:1.The mechanism for MtPAR regulating PA biosynthesis and its application in alfalfa:(1)The MtPAR-transgenic Medicago hairy roots had increased PAs,decreased isoflavonoids,and almost non-changed anthocyanin contents,as compared to GUS control.The expression levels of biosynthetic genes showed the same trend with metabolome analysis results.To understand how MtPAR affected isoflavone synthesis,soybean hairy roots overexpressing MtPAR were generated for detection of the changes in metabolome and transcriptome.By using yeast one-hybrid technique,we confirmed that MtPAR could bind to the promoter of IFS2 gene,and suppress IFS2 expression,eventually resulting in reduced isoflavone production.The tt2 mutant seeds displayed a pale yellow color,due to lack of PA production in seed coat,compared to the brown color of wild-type seeds.Expression of MtPAR gene in tt2 mutant plants restored the seed color phenotype.The overexpression of MtPAR in wild-type plants increased the total seed PA content by about 22% compared with wild-type,suggesting that MtPAR shares similar function with At TT2.Further studies revealed that MtPAR interacted with Mt TT8 and Mt WD40-1 to form a complex,which can activate ANR promoter and regulate anthocyanin and PA biosynthesis.Thus,we concluded that MtPAR may reduce the flavonoid precursor flux into isoflavone pathway but direct more precursor flows into anthocyanin and PA biosynthesis.(2)MtPAR improved the performance of metabolic engineering of PA production in alfalfa.Studies have shown that more PA in alfalfa leaves and stem can protect ruminants such as cows and sheeps from leathal pasture bloat,which causes the loss of millions of dollars a year worldwide.We demonstrated that that dual regulation of MtPAR in flavonoid and isoflavone biosynthesis pathways,and tried to further enhance PA production in alfalfa by using the mechanisms.For that,we co-expressed an anthocyanidin specific regulator Mt LAP1 with MtPAR to boost the cyanidin precursor supplies.We then detected a significant amount of PAs in aerial part of transgenic alfalfa than that in the control.This study laid the foundation for breeding of new alfalfa variety free of leathal pasture bloat.2.Metabolic regulatory mechanism of flavonoids and isoflavones in soybean:By analyzing the correlation between Gm ANS or Gm ANR genes expression patterns with these for many transcription factors in a transcriptomic analyses,we identified a MYB type transcription factor GmMYB115 that is involved in regulating anthocyanin and PA biosynthesis in soybean.Overexpression of GmMYB115,the closet MtPAR homolog gene from soybean,could partly restored the seed color phenotype of tt2 mutant seeds,and increased the total PA content in wild-type Arabidopsis seeds.GmMYB115 can replace At TT2 to interact At TT8 and At TTG1 proteins to form a MBW ternary complex.Overexpression of GmMYB115 promoted accumulation of anthocyanins in tobacco petals,resulting in the dark red color of petals in transgenic tobacco.Corresdondingly,we also detected the enhanced tobacco ANS and ANR gene expression.Further study disclosed that the ternary MBW complex in soybean,including Gn MYB115,Gm TT8 and Gm TTG1,can activate Gm ANS and Gm ANR genes expression.In transgenic hairy roots overexpressing GmMYB115,the Gm ANS,Gm ANR,Gm LAR genes were significantly up-regulated,although there was no anthocyanidin or PA production was observed in GmMYB115 hairy roots.However,isoflavone biosynthesis-related genes were down regulated meanwhile,leading to the reduction of isoflavone accumulation.GmMYB115 could also bind to IFS2 promoter to suppress IFS2 expression,just like MtPAR.GmMYB115 may thus compete with isoflavone activators to bind the IFS2 promoter.Transcriptome analysis showed that overexpression of GmMYB115 significantly regulated several repressor and activator transcription factor genes.It will be of great interest to further investigate the functions of these downstream activators or repressors in regulation of flavonoid and isoflavone biosynthesis.In a future plan,we will further characterize these repressor and activator genes in soybean hairy root to test their effects on biosynthesis of isoflavones and flavonoids.This will provide insights into our understanding of competition between flavonoid and isoflavone biosynthesis through transcription regulatory mechanism. |
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