Functional Identification And Interaction Analysis Of Flower Development-related ECE And WUS TFs In Chrysanthemum Morifolium

The complex inflorescence of Chrysanthemum morifolium,capitulum,is often comprised of zygomorphic female ray florets and actinomorphic bisexual disc florets.Different flower symmetry and sex expression in ray and disc florets are the main differences between the two florets,but their molecular regulatory mechanisms are not clear yet.It has been proved that regulators of flower symmetry are also involved in the development of reproductive organs,especially in stamens.Therefore,studying the floret development of chrysanthemum from the aspect of flower symmetry will help to clarify the molecular mechanism of capitulum morphogenesis,contribute to a further understanding of the gynomonoecious sexual system in chrysanthemum,and provide a theoretical basis for the enrichment of flower types of chrysanthemum through molecular breeding.Flower symmetry-related ECE(CYC/TB1)and reproductive organ development-related WUSCHEL(WUS)transcription factor genes were selected through our transcriptome analysis in chrysanthemum and CYC2 clade ECE genes have been preliminary studied by our research group.In this study,CYC1 and CYC3 clade ECE genes and WUS were amplified from C.morifolium by RT-PCR and RACE,and their expression patterns were then compared by qPCR.Also,their functions were analyzed by overexpression in Arabidopsis thaliana and C.morifolium.On this basis,yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)assays were performed to comprehensively analyze the protein-protein interactions between ECE and WUS transcription factors.Moreover,the promoter sequence of a key CYC3 clade ECE gene was cloned by genome walking and yeast one-hybrid(Y1H)assays were conducted to study the up-downstream regulatory relationship.The main results of this study are listed as follow:1.Two CYC1 clade and three CYC3 clade ECE genes were isolated from C.morifolium 'Fen Ditan'.These genes were named CmCYCla-lb and CmCYC3a?3c,respectively.Among them,CmCYC1b has two transcripts,CmCYC1b-1 and CmCYC1b-2.2.Analysis of the expression patterns of ECE genes indicated that CmCYC2 genes are key regulators of floret symmetry.Among them,CmCYC2c and CmCYC2e were primary factors in determination and maintenance of ray floret identity,and CmCYC1b and CmCYC2d may have great influences on organ fusion of ray petals.In addition,CmCYC1 and CmCYC3 genes may also be involved in the regulation of floret symmetry and stamen development,as well as the promotion of ray petal fusion.3.Overexpression of CmCYCl and CmCYC3 in A.thaliana led to fewer stamens with shifts in flower symmetry,suggesting that CmCYCl and CmCYC3 are the major regulatory genes in stamen development during the development of capitulum and also participate in the regulation of floret symmetry.Moreover,overexpression of CmCYC3a significantly inhibits vegetative growth and delays flowering.In addition,overexpression of CmCYC1a in A.thaliana increased the number of branches and attenuated apical dominance,which was completely opposite to other CmCYC1 and CmCYC3 genes.4.A protein-protein interaction mode between chrysanthemum ECE transcription factors was proposed.It is speculated that CmCYC2 are the main regulators of floret symmetry,while CmCYCl and CmCYC3 regulate stamen development and participate in the regulation of floret symmetry.All the three clades of ECE transcription factors take part in the regulation of floret symmetry and stamen development through protein-protein interactions,and ultimately affect the morphogenesis of the capitulum.5.CmWUS was isolated from C.morifolium 'Fen Ditan',which was highly expressed in the reproductive organs and disc florets.Overexpression of CmWUS in A.thaliana led to shifts in flower symmetry,fewer stamens and shorter siliques.In addition,CmWUS could directly interact with CmCYC2d and CmCYC3a,and bind to the cis-acting element TTAATGG(or TAAT)in the promoter sequence of CmCYC3a and activate the expression of resistance gene in yeast,indicating that CmWUS and ECE transcription factors are both involved in the regulation of floret symmetry and floral organ development.This study deeply analyzed the functions of flower development-related ECE and WUS transcription factors in C.morifolium,and established a protein-protein interaction network between ECE and WUS,which provided a theoretical basis for molecular breeding to enrich flower types of chrysanthemum.