| Proanthocyanidins(PAs),also known as condensed tannins,are the most important specialized metabolites,which can help plants resist biotic and abiotic stresses.As powerful antioxidants,PAs can provide multiple beneficial effects for human health and crop quality.Although PAs are synthesized via the general flavonoid pathway and share most biosynthetic steps with anthocyanins,there must be a fine regulatory network that controls their synthesis and accumulation respectively.At present,studies on PAs mainly focus on the vegetative organs and fruits of dicotyledonous plants,few reports on the regulation of PAs synthesis have been focused on monocotyledonous plants or flowers.Freesia hybrida,a monocotyledonous genus of herbaceous perennial flowering plants,is a good material for studying the metabolism of flavonoids in plants.Previously,a MYB regulator named as FhMYB5 was isolated and confirmed to function in the general flavonoid pathway,which contributed to the regulation of anthocyanin and PA accumulations in Freesia.In this study,four TT2-type R2R3MYB transcription factors phylogenetically clustered with At TT2 were further isolated from the flowers of"Red River~?",designated as FhMYBPAs.In order to verify their regulatory roles in the biosynthesis of PAs in Freesia hybrida,a series of experiments have been performed and the main results were summarized as follows:1.Cloning and bioinformatic analysis of FhMYBPAsBased on the transcriptomic database,four TT2-type R2R3 MYB transcription factors regulating the PA pathway were isolated through in situ TBLASTN analysis using At TT2 as probe.Analyses of the primary structure revealed that the R2R3repeat region of FhMYBPAs is highly homologous to other plant MYB factors and contains the residues of a conserved signature sequence[D/E]Lx2[R/K]x3Lx6Lx3R,which is responsible for the interaction with b HLH proteins.Meanwhile,the C-terminal conserved motifs in FhMYBPAs were also found,indicating the four FhMYBPAs were grouped into PA-clade2.2.Expression patterns of FhMYBPAsTwo groups of expression patterns were observed for the four TT2-type MYB regulatory genes aforementioned:FhMYBPA1,FhMYBPA2,and FhMYBPA4,which were clustered together and separated with FhMYBPA3,showed an identical expression profile significantly correlated with FhLAR,the gene involved in the biosynthesis of catechin-derived proanthocyanidins.In contrast,the expression of FhMYBPA3 was found to be synchronous to FhANR,which is responsible for epicatechin-derived proanthocynidins biosynthesis.Therefore,it is reasonable to deduce that FhMYBPAs genes in Freesia might function divergently to control the biosynthesis of the two categories of proanthocyanidins.3.Heterologous expression of FhMYBPAs in tobacco plantsThe four FhMYBPAs genes were successfully ectopically expressed in tobacco plants,hyper-accumulation of proanthocyanidins was observed in individual transgenic lines,which was consistent with the up-regulation of the expressions of Nt DFR,Nt ANS,Nt LAR,Nt ANR genes.Moreover,in accordance with TT2-type proteins from model plant as well as other extensively studied plant species,FhMYBPAs were also found to be interacted with b HLH proteins participating in the MBW complex,and thus control the expression of the PA biosynthetic genes.In conclusion,it can be speculated that TT2-type MYB genes might functional in the biosynthesis of proanthocyanidins in Freesia hybrida.To our knowledge,this is the first reports of the FhMYBPAs genes isolated from flowers of Freesia and even in Liliales plants,results in this study will not only provide new insights into the transcriptional regulation of proanthocyanidin biosynthesis in monocot plants,but also pave the way to dissect the crosstalk networks controlling multiple flavonoids biosynthesis which are simultaneously accumulated in the same plant organs with diverse patterns. |
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