CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum.However,the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear,which greatly restricts design of molecular breeding in chrysanthemum.Here,a chrysanthemum SG7 R2R3 MYB transcription factor(TF),CmMYB3-like,was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage.Quantitative real time PCR(qRT-PCR)assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage.Genetic transformation of chrysanthemum and dual-luciferase assays in N.benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly.However,overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols.Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation,but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage.Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

CmWRKY6-1-CmWRKY15-like transcriptional cascade negatively regulates the resistance to fusarium oxysporum infection in Chrysanthemum morifolium

Chrysanthemum Fusarium wilt is a soil-borne disease that causes serious economic losses to the chrysanthemum industry.However,the molecular mechanism underlying the response of chrysanthemum WRKY to Fusarium oxysporum infection remains largely unknown.In this study,we isolated CmWRKY6–1 from chrysanthemum‘Jinba’and identified it as a transcriptional repressor localized in the nucleus via subcellular localization and transcriptional activation assays.We found that CmWRKY6–1 negatively regulated resistance to F.oxysporum and affected reactive oxygen species(ROS)and salicylic acid(SA)pathways using transgenic experiments and transcriptomic analysis.Moreover,CmWRKY6–1 bound to the W-box element on the CmWRKY15-like promoter and inhibited its expression.Additionally,we observed that CmWRKY15-like silencing in chrysanthemum reduced its resistance to F.oxysporum via transgenic experiments.In conclusion,we revealed the mechanism underlying the CmWRKY6–1–CmWRKY15-like cascade response to F.oxysporum infection in chrysanthemum and demonstrated that CmWRKY6–1 and CmWRKY15-like regulates the immune system.

Interaction between MAPKs and MKPs in hexaploid chrysanthemum illuminates functional paralogue diversification in polyploids

Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.

B Class Floral Homeotic Genes are Involved in the Petal Identity and Flower Meristem Determinations in Chrysanthemum morifolium

Chrysanthemum morifolium,an ornamental crop with diverse forms of inflorescence,is a good model for studying flower development in Asteraceae.However,the genetic background is complex and the mechanisms of regulating flower development are still unclear.Here,we identified two natural mutant lines of chrysanthemum and named them M1 and M2 according to the severity of the phenotype.Both lines showed defects in petal identity,and the petals of the M1 line had a mild phenotype:partially loss of petal identity and conversion of petals into green,leaf-like organs.The M2 line had severe phenotypes:in addition to severe petal defects,secondary inflorescences were produced in the capitulum to replace the normal ray and disc florets,which indicated a transformation of a flower meristem into an inflorescence meristem.Transcriptome sequencing of WT and M2 inflorescences was performed and found altered expression of floral organ development A,B and E class genes,where B and E class genes were significantly down-regulated.qRT-PCR analysis in both M1 and M2 lines revealed that the expression of three chrysanthemum class B genes CmAP3.1,CmAP3.2 and CmPI,was negatively correlated with phenotypic severity.This suggests that class B genes in chrysanthemum not only have conserved functions in determining petal identity but also were involved in the determinacy of the flower meristem.This study provides insights into the functions of class B genes in flower development,and is informative for dissecting the molecular mechanisms of flower development in chrysanthemum.

Brassinosteroid Biosynthetic Gene CmDWF4 Regulates Bud Outgrowth in Chrysanthemum morifolium

Brassinosteroids(BRs),a class of steroid phytohormones,play a critical role in plant growth and development.The DWF4 gene encodes a cytochrome P450 enzyme(CYP90B1),which is considered a rate-limiting enzyme in BR biosynthesis.Here,we identified a homologous gene of DWF4 in chrysanthemum,CmDWF4.This gene was predicted to encode 491 amino acid residues with a molecular weight of 56.2 kDa and an isoelectric point(pI)of 9.10.Overexpression of CmDWF4 in chrysanthemum was found to significantly increase growth rate,number,and length of lateral buds.Transcriptome analysis showed that multiple xyloglucan endotransglycosylase/hydrolase(XTH)family encoding genes associated with cell wall modification were up-regulated in CmDWF4-overexpressing lines.qRT-PCR assay confirmed the up-regulation of CmXTH6,CmXTH23,and CmXTH28 in CmDWF4-overexpression line.Overall,this work establishes a mechanism by which BR biosynthetic gene CmDWF4 promotes lateral bud outgrowth in chrysanthemum,possibly through regulating cell elongation and expansion.

Comparative Non Participating Transcriptome Analysis Response to Low Phosphorus by CmPht1;2 in Chrysanthemum

Chrysanthemum morifolium Ramat is one of the four major cut flowers in the world.Pht1 family is focus on the uptake and transport of phosphate in plants.In our previous studies,CmPht1;2 overexpression line(Oe1)had higher phosphate contents both in roots and shoots,and its root development was significantly enhanced than wild type(WT)at low phosphorus conditions in chrysanthemum.Metabolomics analysis showed that several metabolites had a change in pyruvate metabolism and tricarboxylic acid(TCA)cycle pathway.To explore the gene difference expression and the change of metabolic pathway between CmPht1;2-Oe1 and WT,we conducted the transcriptome analysis.A total of 617,681 and 207,271 unigenes were obtained from roots and shoots,respectively.They were classified into biological process,cellular component and molecular function by Gene Ontology(GO).In addition,450 different expression genes(DEGs)were found in the roots after 2 d treatment,and 1,787 DEGs were identified in shoots after 7 d treatment under LP condition between Oe1 and WT.From the top 20 pathways of DEGs assigned by Kyoto Encyclopedia of Genes and Genomes(KEGG),TCA cycle and pyruvate metabolism pathways mostly affected by overexpression of CmPht1;2 attracted our attention.This research will be helpful for elucidating the mechanism of effects by CmPht1;2 overexpression on growth,development and stress tolerance.

江苏省植物细胞遗传学研究回顾与展望

20世纪初“遗传的染色体学说”的提出和证明标志着细胞遗传学交叉学科建立,伴随相关学科的发展,20世纪60年代末期细胞遗传学又与分子遗传学相结合,建立发展了分子细胞遗传学交叉学科。分子细胞遗传学以DNA分子原位杂交技术为核心,不断拓展应用领域,为生命科学研究提供了直观、高效的技术手段。原位杂交技术与基因组、细胞生物学等技术结合,被广泛应用于人类、动物、植物的起源、进化、驯化等基础研究和远缘杂交、染色体工程等应用研究。通过形象地展示DNA、RNA、蛋白质在细胞中的实际位置,揭示DNA序列之间的实际位置和顺序、亲缘物种间的进化关系和结构重排、基因组拼接序列的质量、转录水平RNA和翻译水平蛋白质的位置和数量变化等。江苏省遗传学会会员单位南京农业大学、扬州大学、南京林业大学、江苏师范大学、徐淮地区农科院等自20世纪中期开展细胞遗传学理论技术研究,伴随学科发展不断创新,建立了较完善的分子细胞遗传技术体系,并成功应用于开展植物系统进化、远缘杂交、染色体工程、基因组学等研究,取得了一批研究成果。本文将主要综述江苏省在该领域取得的重要进展,并展望未来发展方向。

Current achievements and future prospects in the genetic breeding of chrysanthemum:a review

Chrysanthemum(Chrysanthemum morifolium Ramat.)is a leading flower with applied value worldwide.Developing new chrysanthemum cultivars with novel characteristics such as new flower colors and shapes,plant architectures,flowering times,postharvest quality,and biotic and abiotic stress tolerance in a time-and cost-efficient manner is the ultimate goal for breeders.Various breeding strategies have been employed to improve the aforementioned traits,ranging from conventional techniques,including crossbreeding and mutation breeding,to a series of molecular breeding methods,including transgenic technology,genome editing,and marker-assisted selection(MAS).In addition,the recent extensive advances in high-throughput technologies,especially genomics,transcriptomics,proteomics,metabolomics,and microbiomics,which are collectively referred to as omics platforms,have led to the collection of substantial amounts of data.Integration of these omics data with phenotypic information will enable the identification of genes/pathways responsible for important traits.Several attempts have been made to use emerging molecular and omics methods with the aim of accelerating the breeding of chrysanthemum.However,applying the findings of such studies to practical chrysanthemum breeding remains a considerable challenge,primarily due to the high heterozygosity and polyploidy of the species.This review summarizes the recent achievements in conventional and modern molecular breeding methods and emerging omics technologies and discusses their future applications for improving the agronomic and horticultural characteristics of chrysanthemum.

Comparative Analysis of Leaf Trichomes, Epidermal Wax And Defense Enzymes Activities in Response to Puccinia horiana in Chrysanthemum and Ajania Species

White rust caused by Puccinia horiana is a destructive disease of chrysanthemum plants.To better understand the resistance mechanisms of composite species to this disease,the leaf cuticular traits,antioxidant and defensive enzymes activities of immune(Chrysanthemum makinoi var.wakasaense)and highly susceptible(Ajania shiwogiku var.kinokuniense)species were compared.Trichome density of two species was markedly different,negatively associated with plant resistance to P.horiana.Total wax load in C.makinoi var.wakasaense was two times more than that in A.shiwogiku var.kinokuniense.The wax composition in immune one was abundant in esters and primary alcohols.Superoxide dismutase(SOD,EC 1.15.1.1),peroxidase(POD,EC 1.11.1.7),polyphenoloxidase(PPO,EC 1.14.18.1 or EC 1.10.3.2)and phenylalanine ammonia lyase(PAL,EC 4.3.1.5)activitieswere investigated.In C.makinoi var.wakasaense,the activity of SOD and POD increased rapidly after inoculation,whichmight be non-host induced reactive oxygen species(ROS)activated antioxidant enzymes,however SOD and POD remained a low and steady level in the highly susceptible one after inoculation.Quick increase in PPO activities after inoculation was observed in both species,however it remained higher in C.makinoi var.wakasaense at the late period of inoculation.PAL in C.makinoi var.wakasaense was induced after pathogen inoculation,but not in A.shiwogiku var.kinokuniense,suggesting that these two enzymes might contribute to the resistance to P.horiana.

Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums

Chrysanthemums are sensitive to waterlogging stress,and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding.To understand the genetic basis of waterlogging tolerance(WT)in chrysanthemums,we performed a genome-wide association study(GWAS)using 92,811 single nucleotide polymorphisms(SNPs)in a panel of 88 chrysanthemum accessions,including 64 spray cut and 24 disbud chrysanthemums.The results showed that the average MFVW(membership function value of waterlogging)of the disbud type(0.65)was significantly higher than that of the spray type(0.55)at P<0.05,and the MFVW of the Asian accessions(0.65)was significantly higher than that of the European accessions(0.48)at P<0.01.The GWAS performed using the general linear model(GLM)and mixed linear model(MLM)identified 137 and 14 SNP loci related to WT,respectively,and 11 associations were commonly predicted.By calculating the phenotypic effect values for 11 common SNP loci,six highly favorable SNP alleles that explained 12.85—21.85%of the phenotypic variations were identified.Furthermore,the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified(r2=0.45;P<0.01).A major SNP locus(Marker6619-75)was converted into a derived cleaved amplified polymorphic sequence(dCAPS)marker that cosegregated with WT with an average efficiency of 78.9%.Finally,four putative candidate genes in the WT were identified via quantitative real-time PCR(qRT-PCR).The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection(MAS)in chrysanthemum WT breeding programs.

A novel transcription factor CmMYB012 inhibits flavone and anthocyanin biosynthesis in response to high temperatures in chrysanthemum

Flavones are among the major colorless pigments synthesized through branches of the flavonoid pathway in plants.However,due to the absence of a gene encoding flavone synthase(FNS)in the model plant Arabidopsis thaliana species,the regulatory mechanism of FNS-catalyzed flavone biosynthesis has rarely been studied in plants.Here,it was found that flavones play a predominant role in the elimination of excess reactive oxygen species(ROS)at high temperatures in colorless plant organs.A novel atypical subgroup 7(SG7)R2R3-MYB transcription factor,CmMYB012,was found to be induced in response to prolonged high temperatures and to inhibit flavone biosynthesis by directly regulating CmFNS.Moreover,CmMYB012 was also found to inhibit anthocyanin biosynthesis by suppressing the expression of CmCHS,CmDFR,CmANS,and CmUFGT.CmMYB012 overexpression exerted a negative in fluence on plant fitness and pink flower color formation,while CmMYB012 suppression had the opposite effect in response to high temperatures.Our findings provide new insights into the mechanisms by which high temperatures regulate the metabolism of flavones and anthocyanins to affect plant fitness and flower color formation.

Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium

As the junction of floral development pathways,the FLOWERING LOCUS T(FT)protein called‘florigen’plays an important role in the process of plant flowering through signal integration.We isolated four transcripts encoding different isoforms of a FT orthologous gene CmFTL1,from Chrysanthemum morifolium cultivar‘Jimba’.Sequence alignments suggested that the four transcripts are related to the intron 1.Expression analysis showed that four alternative splicing(AS)forms of CmFTL1 varied depending on the developmental stage of the flower.The functional complement experiment using an Arabidopsis mutant ft-10 revealed that the archetypal and AS forms of CmFTL1 had the function of complementing late flower phenotype in different levels.In addition,transgenic confirmation at transcript level showed CmFTL1 and CmFTL1ast coexist in the same tissue type at the same developmental stage,indicating a post-transcriptional modification of CmFTL1 in Arabidopsis.Moreover,ectopic expression of different AS forms in chrysanthemum resulted in the development of multiple altered phenotypes,varying degrees of early flowering.We found that an alternative splicing form(CmFTL1-astE134)without the exon 2 lacked the ability causing the earlier flower phenotype.The evidence in this study indicates that complex alternative processing of CmFTL1 transcripts in C.morifolium may be associated with flowering regulation and hold some potential for biotechnical engineering to create early-flowering phenotypes in ornamental cultivars.

Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin

MYB transcription factors are widely involved in the development of and physiological processes in plants.Here,we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15,a homologous gene of AtMYB15.It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements,which usually exist in the promoter of lignin biosynthesis genes.Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids.Additionally,the content of lignin and the expression of several lignin biosynthesis genes increased.In summary,the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

CmFTL2 is involved in the photoperiod- and sucrose-mediated control of flowering time in chrysanthemum

The chrysanthemum genome harbors three FT-like genes:CmFTL1 and CmFTL3 are thought to act as regulators of floral induction under long-day(LD)and short-day(SD)conditions,respectively,whereas the function of CmFTL2 is currently unclear.The objective of the present research was to explore the function of CmFTL2 in the determination of flowering time of the photo-insensitive chrysanthemum cultivar‘Floral Yuuka’,both in response to variation in the photoperiod and to the exogenous provision of sucrose.Spraying leaves of‘Floral Yuuka’plants with 50 mM sucrose accelerated flowering and increased the level of CmFTL2 transcription in the leaf more strongly than either CmFTL1 or FTL3 under both long and SD conditions.Transcription profiling indicated that all three CmFTL genes were upregulated during floral induction.The relationship of the CmFTL2 sequence with that of other members of the PEBP family suggested that its product contributes to the florigen rather than to the anti-florigen complex.The heterologous expression of CmFTL2 in the Arabidopsis thaliana ft-10 mutant rescued the mutant phenotype,showing that CmFTL2 could compensate for the absence of FT.These results suggest that CmFTL2 acts as a regulator of floral transition and responds to both the photoperiod and sucrose.

A temporal gene expression map of Chrysanthemum leaves infected with Alternaria alternata reveals different stages of defense mechanisms

Chrysanthemum(Chrysanthemum morifolium)black spot disease(CBS)poses a major threat to Chrysanthemum cultivation owing to suitable climate conditions and current lack of resistant cultivars for greenhouse cultivation.In this study,we identified a number of genes that respond to Alternaria alternata infection in resistant and susceptible Chrysanthemum cultivars.Based on RNA sequencing technology and a weighted gene coexpression network analysis(WGCNA),we constructed a model to elucidate the response of Chrysanthemum leaves to A.alternata infection at different stages and compared the mapped response of the resistant cultivar‘Jinba’to that of the susceptible cultivar‘Zaoyihong’.In the early stage of infection,when lesions had not yet formed,abscisic acid(ABA),salicylic acid(SA)and EDS1-mediated resistance played important roles in the Chrysanthemum defense system.With the formation of necrotic lesions,ethylene(ET)metabolism and the Ca^(2+)signal transduction pathway strongly responded to A.alternata infection.During the late stage,when necrotic lesions continued to expand,members of the multidrug and toxic compound extrusion(MATE)gene family were highly expressed,and their products may be involved in defense against A.alternata invasion by exporting toxins produced by the pathogen,which plays important roles in the pathogenicity of A.alternata.Furthermore,the function of hub genes was verified by qPCR and transgenic assays.The identification of hub genes at different stages,the comparison of hub genes between the two cultivars and the highly expressed genes in the resistant cultivar‘Jinba’provide a theoretical basis for breeding cultivars resistant to CBS.

CmBES1 is a regulator of boundary formation in chrysanthemum ray florets

Chrysanthemum(Chrysanthemum morifolium)is an ideal model species for studying petal morphogenesis because of the diversity in the flower form across varieties;however,the molecular mechanisms underlying petal development are poorly understood.Here,we show that the brassinosteroid transcription factor BRI1-EMS-SUPPRESSOR 1(CmBES1)in chrysanthemum(C.morifolium cv.Jinba)is important for organ boundary formation because it represses organ boundary identity genes.Chrysanthemum plants overexpressing CmBES1 displayed increased fusion of the outermost ray florets due to the loss of differentiation of the two dorsal petals,which developed simultaneously with the ventral petals.RNA-seq analysis of the overexpression lines revealed potential genes and pathways involved in petal development,such as CUP-SHAPED COTYLEDON(CUC2),CYCLOIDEA 4(CYC4),genes encoding MADS-box transcription factors and homeodomain-leucine zippers(HD-Zips)and auxin pathway-related genes.This study characterizes the role of CmBES1 in ray floret development by its modulation of flower development and boundary identity genes in chrysanthemum.

Chrysanthemum CmWRKY53 negatively regulates the resistance of chrysanthemum to the aphid Macrosiphoniella sanborni

Chrysanthemum is frequently attacked by aphids,which greatly hinders the growth and ornamental value of this plant species.WRKY transcription factors play an important role in the response to biotic stresses such as pathogen and insect stresses.Here,chrysanthemum CmWRKY53 was cloned,and its expression was induced by aphid infestation.To verify the role of CmWRKY53 in resistance to aphids,CmWRKY53 transgenic chrysanthemum was generated.CmWRKY53 was found to mediate the susceptibility of chrysanthemum to aphids.The expression levels of secondary metabolite biosynthesis genes,such as peroxidase-and polyphenol oxidase-encoding genes,decreased in CmWRKY53-overexpressing(CmWRKY53-Oe)plants but dramatically increased in chimeric dominant repressor(CmWRKY53-SRDX)plants,suggesting that CmWRKY53 contributes to the susceptibility of chrysanthemum to aphids,possibly due to its role in the regulation of secondary metabolites.

Regulation of flowering time in chrysanthemum by the R2R3 MYB transcription factor CmMYB2 is associated with changes in gibberellin metabolism

The switch from vegetative growth to reproductive growth is a key event in the development of a plant.Here,the product of the chrysanthemum gene CmMYB2,an R2R3 MYB transcription factor that is localized in the nucleus,was shown to be a component of the switching mechanism.Plants engineered to overexpress CmMYB2 flowered earlier than did wild-type plants,while those in which CmMYB2 was suppressed flowered later.In both the overexpression and RNAi knockdown plants,a number of genes encoding proteins involved in gibberellin synthesis or signaling,as well as in the response to photoperiod,were transcribed at a level that differed from that in the wild type.Both yeast two-hybrid and bimolecular fluorescence complementation assays revealed that CmMYB2 interacts with CmBBX24,a zinc-finger transcription factor known to regulate flowering by its influence on gibberellin synthesis.

Overexpression of the CmJAZ1-like gene delays flowering in Chrysanthemum morifolium

Chrysanthemum(Chrysanthemum morifolium)is one of the four major cut-flower plants worldwide and possesses both high ornamental value and cultural connotation.As most chrysanthemum varieties flower in autumn,it is costly to achieve annual production.JAZ genes in the TIFY family are core components of the jasmonic acid(JA)signaling pathway;in addition to playing a pivotal role in plant responses to defense,they are also widely implicated in regulating plant development processes.Here,we characterized the TIFY family gene CmJAZ1-like from the chrysanthemum cultivar‘Jinba’.CmJAZ1-like localizes in the nucleus and has no transcriptional activity in yeast.Tissue expression pattern analysis indicated that CmJAZ1-like was most active in the root and shoot apex.Overexpressing CmJAZ1-like with Jas domain deletion in chrysanthemum resulted in late flowering.RNA-Seq analysis of the overexpression lines revealed some differentially expressed genes(DEGs)involved in flowering,such as the homologs of the flowering integrators FT and SOC1,an FUL homolog involved in flower meristem identity,AP2 domain-containing transcription factors,MADS box genes,and autonomous pathway-related genes.Based on KEGG pathway enrichment analysis,the differentially transcribed genes were enriched in carbohydrate metabolic and fatty acid-related pathways,which are notable for their role in flowering in plants.This study preliminarily verified the function of CmJAZ1-like in chrysanthemum flowering,and the results can be used in molecular breeding programs aimed at flowering time regulation of chrysanthemum.

CmRCD1 represses flowering by directly interacting with CmBBX8 in summer chrysanthemum

The CmBBX8-CmFTL1 regulatory module is a key determinant in the transition from vegetative growth to reproductive development in summer-flowering chrysanthemum.However,the detailed regulatory mechanism of CmBBX8-mediated flowering remains elusive.In this study,we revealed that RADICAL-INDUCED CELL DEATH 1(CmRCD1)physically associated with CmBBX8 through bimolecular fluorescence complementation(BiFC),pulldown and Coimmunoprecipitation(CoIP)assays.Furthermore,the RCD1-SRO1-TAF4(RST)domain of CmRCD1 and the B-box of CmBBX8 mediated their interaction.In addition,Luciferase(LUC)assays and electrophoretic mobility shift assay(EMSAs)showed that CmRCD1 repressed the transcriptional activity of CmBBX8 and interfered with its binding to the CmFTL1 promoter,thereby leading to delayed flowering in the summer chrysanthemum‘Yuuka’.These results provide insight into the molecular framework of CmRCD1-CmBBX8-mediated flowering in chrysanthemum.