Mechanisms For Transcriptional Regulation Of Anthocyanin Biosynthesis By MYB-bHLH-WD40 In Paeonia Suffruticosa

Tree peony(Paeonia suffruticosa Andrews)is an important traditional ornamental and medicinal plant that is indigenous to China;it belongs to the family Paeoniaceae and is valued for its large,showy and colorful flowers.Flower color is an essential ornamental trait for ornamental plants.Flavonoids are secondary metabolites that determine flower colors,which confer a wide spectrum of color that includes pale yellow,scarlet,red,magenta,violet,and blue to flowers.In generally,transcriptional regulation of the structural genes is controlled by MYB-bHLH(basic helix-loop-helix)-WD40 complex.So far,there are few studies on the transcriptional regulation of flower colouration of tree peony.The tree peony cultivar P.suffruticosa ‘Yuban Bai',‘Zhao fen' and ‘Heihua Kui' were used in this study.Anthocyanins and flavonoids were examined with Ultra-high-performance liquid chromatography–mass spectrometry(UPLC-MS/MS,Waters,Milford,MA,USA).Based on the transcriptome data,the key structural genes in anthocyanin synthesis pathway were screened and candidate MYB/bHLH/WD40 transcription factors may involved in anthocyanin regulation were identified.The mechanisms of these transcription factors regulating anthocyanin biosynthesis in tree peony were discussed in this study.The study aims to provide the basis to reveal the regulation mechanism of flower colouration in tree peony and also provide valuable genes for flower colour molecular breeding of tree peony.The main results were as follows:1.The Ultrahigh Performance Liquid Chromatogram(UPLC-DAD-ESI-MS/MS)was applied to analyze the anthocyanins and flavonoids components of tree peony cultivars 'Zhao Fen','Yuban Bai' and 'Heihua Kui'.The results showed that no anthocyanins or derivatives were detected in P.suffruticosa ‘Yuban Bai'(white).Two types of anthocyanin were detected in the petals of P.suffruticosa ‘Zhao Fen',which were Pg3G5 G and Pn3G5 G.Five types of anthocyanins were detected in the petals of P.suffruticosa ‘Heihua Kui' as follows: Pn3G5 G,Cy3G5G,Cy3 G,Pn3G and Pg3G5 G.Eighteen kinds of flavonoids and flavonol aglycones were detected in the three cultivars.It is concluded that the existence of flavonoids and flavonol aglycones plays an important role in the color development of white peony.The Quantitative analysis results on the pigment composition of P.suffruticosa ‘Heihua Kui' showed that Pn3G5 G,Cy3G5G and Cy3 G were the main pigments during the whole development of petals.The contents of anthocyanin and flavonoid were lower in the stage 1 and increased significantly from stage 1 to stage 2.The contents of anthocyanin and flavonoid were increased from stage 3 to stage 4,but they tended to be stable.2.The transcriptome from petals of tree peony was contructed and sequenced using the Illumina novaseq 6000 platform.After data filtering and sequence assembly,167702 unigenes were generated,with an average length of 707 nt.Approximately 39.83% of the unigenes(66,797)could be annotated based on public protein databases(NR,Swiss-Prot,KEGG,KOG).The DEGs of structural genes involved in anthocyanin biosynthesis were screened out and verified by qRT-PCR.The results indicated that PsF3H?PsF3'H?PsDFR and PsANS may be responsible for the color difference among the three tree peony cultivars.3.We identified 84 PsbHLH genes in the petals of tree peony based on the transcriptome data,named sequentially as PsbHLH1 to PsbHLH84.The phylogenetic relationship between tree peony and Arabidopsis bHLH transcription factors showed that tree peony bHLH genes were devided into 31 subgroups.In phylogenetic analysis,tree peony PsbHLH1/PsbHLH2/PsbHLH3 and Arabidopsis AtbHLH001?AtbHLH002?AtbHLH012 and AtbHLH042 were classified in the same subfamily(the fifth subfamily).The fifth subfamily has been confirmed to be involved in the regulation of anthocyanin and procyanidin synthesis.Sequence alignments of PsbHLH1-3 and those related to anthocyanin biosynthesis in other plants showed three intact functional domains named MIR(the MYB interaction region),bHLH domain and ACT-like.The PsbHLH1 protein with His5-Glu9-Arg13(H-E-R)and Arg10-Arg12 residues have been shown to bind to a variation of the E-box hexanucleotide sequence(E-box: CANNTG,variation: CACGTG)and the DNA backbone,respectively.Different residues in these positions(Asn5-Asp9-Gly13 ? Arg10)were present in PsbHLH2and(His5-Asp9-Lys13 ? Gly10-Arg12)were present in PsbHLH3.The expression of PsbHLH1,PsbHLH2 and PsbHLH3 was prior to the formation of anthocyanin by qRT-PCR,which played a major regulatory role in the upstream of structural genes.Subcellular localization assay showed that three TFs were localized and functioned in the nucleus.4.Based on the transcriptome sequencing(RNA-Seq)data,123 sequences of MYB family proteins,including 60 R2R3-MYB proteins,57 MYB related proteins,four R1R2R3-MYB proteins and two 4R-MYB proteins,were annotated in the study.Besides,R1R2R3-MYB protein and 4R-MYB protein were screened out for the first time.The phylogenetic relationship between tree peony and Arabidopsis MYB transcription factors showed that tree peony MYB genes were devided into 25 subgroups.The research showed that the genes of S4,S6 and S7 subgroup have played an important role in the flavonoid biosynthesis.It was found that PsMYB1 gene of S6 subgroup was the key gene regulating anthocyanin synthesis by qRT-PCR,which was expressed in high level incolored varieties.Sequence alignments of PsMYB1 and PsMYB2 showed that both proteins contained highly conserved motif[DE]Lx2[RK]x3Lx6Lx3R in the R3 domain of N-terminal MYB domain,indicating that they could interact with bHLH protein.Besides,PsMYB1 and PsMYB2 contained highly conserved motif [K/R]P[Q/R]P[Q/R],which involved in anthocyanin synthesis.It is concluded that PsMYB1 and PsMYB2 may be involved in anthocyanin synthesis.In addition,subcellular localization assay showed that two TFs were localized and functioned in the nucleus.5.We identified 38 PsWD40 genes in the petals of tree peony based on the transcriptome data,named sequentially as PsWD40-1 to PsWD40-38.The WD40 transcription factors involved in anthocyanin synthesis in other plants and the WD40 transcription factors in tree peony were used to construct phylogenetic trees.It was concluded that PsWD40-1 and PsWD40-2 might be involved in the biosynthesis of anthocyanin in petals of tree peony.The homologous sequence analysis showed that there were four highly conserved WD motifs in PsWD40-1 and PsWD40-2,and the last two amino acid residues(WD,FD,LD and WE)were highly conserved among different species.In addition,subcellular localization assay showed that two TFs were localized and functioned in the nucleus.6.Yeast two-hybrid(Y2H)and bimolecular fluorescence complementation assays(Bi FC)demonstrated that the PsbHLH1 protein interacted with PsMYB1 and PsWD40-1 protein,whereas PsMYB1 was not interact with PsWD40-1 in tree peony.This conclusion was consistent with the regulation model of MBW complex in apple.To explore the regulatory mechanism of transcription factors on anthocyanin biosynthetic genes in tree peony,we cloned the promoter sequences of eight structural genes(PsCHS,PsCHI,PsF3 H,PsF3'H,PsFLS,PsFNS,PsDFR and PsANS)by Genome Walking.The results of promoter elements analysis showed that the putative binding sites for bHLH and MYB transcription factors existed in the promoters of these structural genes,including Myb-binding site(MBS),MYB recognition site(MRE),E-box sequences(CANNTG)and G-box(CACGTG).Yeast one-hybrid(Y1H),a transient dual-luciferase assay and a firefly luciferase complementation assay were performed.In conclusion,these results demonstrated that PsbHLH1 interacts with PsMYB1 and PsWD40-1 in a protein complex and directly activates the expression of PsDFR and PsANS,which was also involved in the anthocyanin biosynthesis in petals of tree peony.