| Clyysanthemum morifolium is a world-renowned Chinese traditional flowers with abundant flower shapes and delicate inflorescence.The inflorescence of the chrysanthemum is composed of both bilaterally symmetrical ray florets and radially symmetrical disc florets.Different composition and distribution of the ray and disc florets contribute to the abundant cultivars of different flower shapes.Despite continuing efforts,the molecular mechanisms underlying regulation of the two floral types and the process of evolution are still unclear so far.CYC(CYCLOIDEA),belonging to CYC2 subfamily of the TCP transcription factors,have been shown critical in controlling flower symmetry or regulating flower-type identity in several angiosperm plant lineages.However,little information about this gene family in C.morifolium has been published.The number,evolution process and roles of these CYC2 genes in regulating floral development in the chrysanthemum are still unknown.Therefore,a total of six CYC2 subfamily genes were amplified from C.morifolium using the RACE-PCR method.The expression patterns of these genes were then compared during early inflorescence development stages and among several different floral heads with various whorls of ray florets.Moreover,the interactions between these CmCYC2 proteins were researched by yeast two-hybrid assay.Several key candidate genes were then further characterized via overexpression in C.lavandulifolium and the Arabidopsis rabidopsis tcp1 mutant.Furthermore,the promoter sequences of several CmCYC2 genes were obtained,then the yeast one-hybrid and dual-luciferase reporter assays were used to study the up-downstream regulate modes of these genes.The main results of this study are listed as follow:1.A total of six CmCYC2 genes,named CmCYC2a-CmCYC2f,were isolated from C.morifolium.A conserved motif analysis showed that all these genes contain the conserved TCP and R domain,and belong to the CYC2 subclade.The phylogenetic analysis placed all the six genes in different clades,suggesting both lineage specific and shared duplications in Asteraceae.Moreover,two transcripts of CmCYC2c were obtained from chrysanthemum and several other closely related species of Chrysanthemum.2.The expression patterns of these genes were compared in two chrysanthemum cultivars and their F1 progenies.All these genes were predominantly expressed in reproductive organs,and barely expressed in vegetative organs,except for CmCYC2a,which was highly expressed in shoot apices.CmCYC2b,CmCYC2c and CmCYC2d were mainly expressed in ray florets.Besides,the expression of CmCYC2c was abundantly in ray florets of different whorls,and dramatically up-regulated in ray florets of the double-ray flowered heads when compared with the expression in the radiate and semi-double ray flowered heads.However,it was barely expressed in the inflorescences of Ajania potaninii and Brachanthemum titovii,which bare few incomplete ray florets.Further,the expression of these genes all peaked at stage ?a,in which the petal primordia of florets began to differentiate,and then decreased gradually.When compared with the other CYC genes in other plant species,the expression patterns of most CmCYC2 genes were both redundant and diverged.3.The results of yeast two-hybrid assay show that heteromeric interaction appeared to occur preferentially among the proteins of CmCYC2c,CmCYC2c-2,CmCYC2d,CmCYC2e and CmCYC2f.Besides,they also exhibited substantial activity for homodimerization.The CmCYC2a and CmCYC2b neither hardly interacted with other CYC2 proteins nor formed homodimers.Subcellular localization analysis revealed that the CmCYC2b,CmCYC2c and CmCYC2d proteins mainly localized into the nucleus of epidermal cells,while the CmCYC2a protein positioned in both cytoplasm and nucleus.The comprehensive analysis of gene expression and protein-protein interaction patterns show that most CmCYC2 proteins can interact with each other in the process of floral organ development,and may be involved in regulating the morphogenesis of ray and disc florets.4.When compared with the control plant lines,the early vegetative growths of the Arabidopsis of tcp1 mutant ectopic expressing CmCYC2 genes were repressed,and the flowering times were also postponed.Moreover,it seems that the size and arrangement of two petals were changed in transgenic lines overexpressing CmCYC2a,CmCYC2b,CmCYC2c and CmCYC2d respectively,making the petal arrangement show bilateral symmetry from the original radial symmetry.The development of one stamen was repressed in the transgenic lines overexpressing CmCYC2e.Furthermore,overexpression of CmCYC2c in C.lavandulifolium led to significant increase in flower numbers and ventral petal ligule length of ray florets.Besides CmCYC2c,the expression of CmCYC2f was also significantly upregulated in transgenic lines.5.The promoter sequences of CmCYC2b,CmCYC2c,CmCYC2d and CmCYC2f were amplified from C.lavandulifolium overexpressing CmCYC2c and C.morifolium respectively.Further promoter analysis led to identification of a putative cis-regulatory element GGNCCCNC,matching the consensus DNA binding site for the TCP transcription factors.This sequence was then used as a bait in yeast one-hybrid assay.The results show that CmCYC2c could bind on it and activate the expression of the downstream AbA resistant gene.In addition,the dual-luciferase transient assay was also carried out to verify the activation ability of protein CmCYC2c.In conclusion,ectopic expression of CmCYC1 subfamily gene in Arabidopsis tcpl mutant lead to the change of petal arrangement and then floral symmetry.Further,CmCYC2c was involved in regulating ray floret identity in the chrysanthemum,by positively regulating the expression of CmCYC2f and interacting with the proteins of CmCYC2c-2,CmCYC2d and CmCYC2e.In this study,the CmCYC2 subfamily genes and their promoters were amplified,and their roles in regulating ray floret development in the chrysanthemum were explored.This study will be useful for genetic manipulation of flower shape in chrysanthemum and hence promote the process of molecular breeding. |
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