Molecular Mechanism Of Anthocyanin Metabolism In Zinnia Elegans

Zinnia elegans is an annual herbaceous flower belonging to the genus Zinnia,family Compositae.Z.elegans is an excellent ornamental plant in flowerbed with numerous colors and great ornamental values.Anthocyanin is one of the essential pigments that affects the petal color in Z.elegans,but the molecular mechanism of anthocyanin metabolism has not been reported.In order to reveal the metabolism mechanism of anthocyanin in Z.elegans,this study employed six cultivars from‘Dreamland’ series with different colors(ivory DI,yellow DY,pink DP,rose DRO,coral DC and red DRE)as plant materials,and color phenotype,pigment composition and its distribution in the ray floret were primarily studied.Based on the determination of the components and contents of anthocyanin in different flower colors and expression patterns of structural genes of anthocyanin metabolism,it was figured out that the differences in sequence and expression patterns of related structural genes were regarded as the influencing factors determining petal coloration of different flower colors.Meanwhile,by screening MYB transcription factors in Z.elegans,ZeMYB9 was confirmed to be involved in the synthesis of different anthocyanin components in Z.elegans from the transcriptional regulatory level.The main results are as follows:1.Ray floret phenotypes of six Z.elegans cultivars with different colors were measured and divided into three groups: white(DI),yellow(DY and DC)and red(DP,DRO and DRE)group.With the development of flower opening,the lightness value in different color cultivars gradually decreased,and the yellowness,redness and chroma values continuously rose.DI had the highest lightness value,while DRE had the lowest.DRO,DRE and DC cultivars showed higher redness value.The yellowness was higher in cultivars from yellow group than those in other cultivars,and DC cultivar had the highest chroma value.2.According to the combination analysis of the color reaction and the freehand section of the ray florets,there was no anthocyanin in DI and DY,and there were a great abundance of carotenoids in the palisade tissues of DY.Anthocyanins were found in the ray florets of DP,DRO,DC and DRE,and anthocyanins were mainly located in the epidermal cells of the ray florets.Both anthocyanins and carotenoids were present in the epidermal cells and palisade tissues of DC and DRE.3.High-performance liquid chromatography was used to determine the components and contents of anthocyanin in ray florets of different color cultivars of Z.elegans.No anthocyanin was detected in DI and DY ray florets,and the total anthocyanin content in DRE was the highest.Anthocyanins in DP and DC were composed mainly of pelargonidin derivatives,accounting for more than 86%.DRO and DRE were colored by both cyanidin and pelargonidin derivatives,and cyanidin derivatives respectively accounted for 55.2% and 61.2% in DRO and DRE.4.Six structural genes involved in anthocyanin synthesis,Ze CHS,Ze CHI,ZeF3 H,ZeF3’H,Ze DFR and Ze ANS,were screened out by sequence analysis from transcriptome sequencing.Based on the analysis of the expression patterns of key genes during the flowering process of Z.elegans,it was revealed that the expression patterns of ZeF3 H and ZeF3’H genes were closely related to anthocyanin accumulation.The expression levels of ZeF3 H gene were pretty low in DI and DY ray florets.Sequence analysis showed that 5 bp sequence was inserted into the ZeF3 H gene transcripts of DI and DY,which resulted in the advance of the termination codon.The expression levels of ZeF3’H gene in the petals of DRE and DRO were significantly higher than those in DP and DC.5.Sequences of 33 ZeMYB genes were screened from Z.elegans transcriptome database.Among them,ZeMYB9,ZeMYB10 and ZeMYB22 genes belonged to Subgroup 6 which was closely related to anthocyanin synthesis.The expression levels of both ZeMYB9 and ZeMYB10 genes were higher in petals than those in phyllaries,stems and leaves.But ZeMYB22 gene was highly expressed in the phyllaries.With the flower opening,the expression of ZeMYB9 gene first increased and then decreased,and the expression of ZeMYB10 gene gradually increased,and the expression of ZeMYB22 gene significantly decreased.The expression level of ZeMYB9 was positively correlated with ZeF3’H gene expression in Z.elegans at different developmental stages,and the transcriptional regulation mechanism of ZeMYB9 was further analyzed.6.The upstream sequence of ZeF3’H gene initiation codon was amplified by FPNI-PCR and the length was 1703 bp.Plant CARE analysis revealed that MYB binding site and other cis-acting elements were present in the ZeF3’H promoter.Dual-luciferase assay showed that ZeMYB9 could significantly activate ZeF3’H promoter.Transient transformation of petunia petals and overexpression of stable transformation of tobacco were used to verify that ZeMYB9 could significantly promote the synthesis and accumulation of anthocyanin in petunia and tobacco corolla.7.In order to investigate the effects of MYB-bHLH complex on anthocyanin synthesis in Z.elegans,a bHLH transcription factor ZeGL3 from Subgroup Ⅲf was found.And it was highly expressed in petals.The interaction between ZeMYB9 and ZeGL3 was verified by bimolecular fluorescence complementation and luciferase complementation assay.Furthermore,the dual-luciferase assay showed that ZeGL3 could activate and promote the ZeF3’H promoter in collaboration with ZeMYB9.Transient overexpression in tobacco leaves showed that only the co-induction of ZeMYB9 and ZeGL3 could significantly promote anthocyanin accumulation in tobacco leaves.It was speculated that ZeMYB9 and ZeGL3 could form a complex to enhance the expression of ZeF3’H and affected the synthesis of anthocyanin components and petal coloration in Z.elegans.