The Molecular Mechanism Of CmERF1 In Regulating Flowering And Flower Development In Chrysanthemum Morifolium

Chrysanthemum(Chrysanthemum morifolium)is one of China's top ten traditional flowers and the world's four major cut flowers,with high ornamental and economic value.Flowering period and flower pattern are important ornamental characters of chrysanthemum.Most chrysanthemum variteties are typical short-day plants and their flowering period directly affects their year-round supply and profits.Chrysanthemum inflorescence is a typical capitulum which is composed of ray and disc florets and the morphology of ray and disk florets directly determines the ornamental quality of the chrysanthemum.The molecular mechanism of flowering and the development of flower in chrysanthemum has been a hot and difficult topic.It is reported that photoperiod pathways and the aging pathway play important roles in the regulation of chrysanthemum flowering time,and CYCLOIDEA2 gene is related to the ray florets development of Asteraceae plants.However,the molecular mechanism of flowering time and flower development in chrysanthemum remains to be further studied.AP2/ERF transcription factors are known to regulate plant growth and development of leaves and flowers.Transcriptome analysis of chrysanthemum under long and short-day conditions showed that the expression level of CmERF1 was significantly induced under short day conditions,however,its roles in regulation of flowering time and so on remained unknown in chrysanthemum.Here,CmERF1 was cloned from chrysanthemum Jinba and the functions and regulatory mechanisms in regulating flowering time and flower development were preliminarily discussed.The main contents and conclusions are as follows:1.CmERFl gene was cloned from chrysanthemum cultivar Jinba,the open reading frame(ORF)is 582 bp and encoding 193 amino acids with a conserved AP2/ERF domain.Phylogenetic trees based on amino acid sequences show that CmERF1 was the most similar to Artemisia annua AaERF3 and closely related to AtERF1 of Arabidopsis thaliana.The expression of CmERF1 is induced by ethylene,the expression level is the lowest in the corolla of ray florets and is the highest in the stamens of disk florets,followed by the pistil of disk florets.It was also found that there was a rhythm in the expression change of CmERF1 under long and short-day,the expression amount reached the peak at 8 h under long-day and at 0 h under short-day condition.CmERFl has no transcriptional activation activity and is located in the nucleus.2.The over-expression vector pMDC43-CmERF1 and RNAi vector pMDC32-amiRNA-CmERF1 were transformed into chrysanthemums Jinba to generate CmERF1 transgenic lines.It was found that the flowering time of CmERF1 overexpressed lines were 7-10 days earlier than that of wild type(WT)plant,but the flowering time of RNAi transgenic lines remained unchanged compared with WT.Using CmERFl as bait protein,yeast two hybridization showed that CmTCP2 is probably interacted with CmERF1.Thereby,CmTCP2 was cloned from chrysanthemum Jinba,its ORF was 1,326 bp in length,encoding 441 amino acids,had a TCP domain,and is closely related to AtTCP2 of Arabidopsis thaliana by phylogenetic analysis.CmTCP2 has no transcriptional activation activity,and is nucleus localized,the expression level of Cm TCP2 is high in leaves and ray florets,and is responsive to the change of circadian rhythm.Direct interaction between CmERF1 and CmTCP2 was verified in vivo and in vitro through yeast two-hybrid,tobacco bimolecular fluorescence complementation(BIFC)assay,and in vitro pull-down experiment.Transcriptome analysis revealed that the flowering time suppressor genes CmCDF2 and CmCDF3 in the photoperiodic pathway were significantly down-regulated in CmERFl overexpressed lines,and the gene encoding florigen of chrysanthemum,CmFTL3,was significantly up-regulated in CmERF1 overexpressed lines,compared with the wild-type.In addition,the expression of circadian genes,CmCCA1 and CmELF3,were significantly down-regulated in CmERFl overexpressed lines.It is speculated that CmERF1 may regulate the expression of photoperiodic pathway genes to regulate flowering time of chrysanthemum.3.Compared with the wild type,the outermost ray florets of CmERFl overexpressed lines formed a tubular structure due to dorsal lobe fusion.The scanning electron microscope observation showed that when the bud diameter was about 5mm,the outermost ray florets of CmERF1overexpressed lines showed obvious dorsal lobe fusion,while the degree of fusion in RNAi lines was lower than that of the wild type.The ratio of length of fused dorsal lobe to the total length of corrolla of outmost ray floret in CmERFl overexpressed lines E12 and E13 were 54.92%and 68.97%respectively,which were significantly higher than those of the wild type(14.60%),and RNAi lines R4 and R12 were 11.80%and 11.11%,respectively.The length of the outermost ray florets of CmERF1 overexpressed lines was shortened by 23 mm,and that in RNAi lines was about 2-5 mm longer than that of the wild type.In situ hybridization showed that CmERF1 was mainly expressed in stamens and pistil of tubular flowers,suggesting that CmERFl might regulate the development of disc florets in chrysanthemum.The expression level of CmCYC2c and CmCYC2e in the outermost ray florets of overexpressed CmERF1 lines was down-regulated,while those in RNAi lines was up-regulated.Subsequently,the promoter sequences of CmCYC2c and CmCYC2e were cloned,and cis-element analysis revealed that there were DRE/CRT cis-acting elements in the promoter sequences of CmCYC2e.ChIP-qPCR analysis preliminarily confirmed that CmERF1 can directly bind to DRE/CRT cis-acting elements present in promoter region of CmCYC2e,but not to CmCYC2c.It is speculated that CmERF1 may inhibit the expression of CmCYC2e gene by directly binding to DRE/CRT cis-acting elements,and indirectly inhibit the expression of CmCYC2c in the outermost ray florets.It is supposed that the ectopic overexpression of CmERF1 may change the expression levels of CmCYC2e and CmCYC2c in the ray florets,consequently,the dorsal lobe of the outermost ray florets can extend and grow,thereby the degree of dosal lobe fusion increases,while the growth of the abdominal lobe is inhibited,thus the outmost ray florets present the shape of tubular like shape.