Molecular Mechanism Of Floral Transition Regulated By The Cryptochromes-mediated Photoperiod Signal Transduction In Chrysanthemum Lavandulifolium

Chrysanthemum(Chrysanthemum X morifolium Ramat.),the famous eastern flower,enjoys a major share of the commercial flower market.Blooming timely is important for annual production of chrysanthemums.Because they are obligate short-day plants,the flowering time of chrysanthemums can be regulated by the use of blackouts or artificial lighting to satisfy the demand for marketable flowers all the year round.However,the method demands much manpower and material resources.Primary influential factor of the flowering time of chrysanthemums at senior phase is the sensitivity to critical photoperiod.The chrysanthemum cultivar,which rapidly responds to short day(SD),could save much cost in practical production.It's vital to explain the molecular mechanism of chrysanthemums responsing to photoperiod signals to complete the floral transition.Oyptochrome(C7RY)genes play vital roles in floral transition specifically responsing to inductive photoperiod conditions based on the molecular mechanism in model plant.Therefore,it is feasible to manipulate the CRY genes involved in floral transition and breed new cultivars of chrysanthemum suitable for annual production.So far,most studies about CRY only focus on the regulation on the expression levels of CONSTANS(CO)protein and FLOWERING LOCUS T(FT)gene.The impact of CRY on other genes in photoperiod pathway has not been clearly characterized.The progress of related researches on the molecular mechanism has been slowly promoted because of the complex genetic background of chrysanthemums.In this study,Chrysanthemum lavandulifolium,a diploid wild chrysanthemum species,was used as a model system for a molecular-genetic study to explore the function and signal transduction mechanisms of CRY.The main results of the present study are the followings.1·Investigating the effects of SD on photomorphogenesis of shoot apex and the expression patterns of FT/TFLl related genes in C.lavandulifolium showed that the shoot apex changed from a conical shape to a dome shape during 0-8 d under SD conditions.Meanwhile,much ClFT1 mRNA was accumulated during 0-8 d SD treatment response to inductive photoperiod.Those results indicated that 0-8 d SD treatment was the key period of floral transition of C.lavandulifolium.Using RT-PCR and 3' RACE method,three CRYs genes(hereafter denominated as CICRY1a,CICRYlb and CICRY2)were isolated from a mixed sample of different C.lavandulifolium organs based on an Illumina/Solexa library.Investigating the effects of SD on the transcription levels of ClCRYs genes in C.lavandulifolium showed that CICRYs mRNA abundance were markedly increased and peaked at 8 d SD treatment.Investigating the effects of different photoperiod conditions on the expression patterns of CICRYs demonstrated more transcripts of CICRYs could accumulate under SD(12 h L/12 h D)compared with that under long day(LD)(16 h L/8 h D)conditions.Those results indicated that ClCRYs genes may be involved in the floral transition of C.lavandulifolium responsing to SD conditions.2.Overexpression of CICRY1a,ClCRY1b or ClCRY2 in wild-type(WT)Arabidopsis resulted in early-flowering phenotype under both LD(16 h L/8 h D)and SD(8 h L/16 h D)conditions with the higher expression levels of FT genes in transgenic Arabidopsis lines.Those results indicated that CICRY1a,CICRY1b and CICRY2 acted as floral enhancers.3.Analysing the flowering phenotype and the expression levels of circadian clock related genes in ClCRYs-overexpression lines and WT plants showed that transgenic chrysanthemums could rapidly response to SD conditions.The transcription levels of ClELF4,CIPRR37,CIZTL,CIFKFl,CICOL1,CICOL2,CICOL4,CICOL5 and CIFT1 were upregulated,and the expression levels of CIPRR73 and CIRVE8 were downregulated in ClCRY1a-overexpressors(CICRYla-OE)plants.The transcription levels of CIELF4,CIPRR37,CIZTL,CIFKF1,ClGI-1,ClGI-2,CICOL1,CICOL2,CICOL4,CICOL5 and ClFT1 were upregulated,and the expression levels of CIRVE8 were downregulated in CICRY1b-OE plants.The transcription levels of CIPRR5,CIZTL,CIFKF1,CIGI-1,ClGI-2,ClCOLl1 CICOL4,CICOL5 and CIFT1 were upregulated,and the expression levels of ClELF3,CIELF4,CILHY,CIPRR73 and CIRVE8 were downregulated in CICRY2-OE plants.Those results indicated that CICRYs could promote the sensitivity of C.lavandulifolium to SD by up-regulating the expression levels of circadian clock related genes,CICOLs and CIFTl.4.A yeast two-hybrid(Y2H)library was screened to identify interacting protein of CICRYs in C.lavandulifolium.Using ClCRY2 as bait,one clone that fulfilled the criteria of interaction with C1CRY2 was isolated and sequenced.The sequence of that clone matches to be a gene RVE8.Subsequently,Y2H assays were conducted to test the interaction of CIRVE8 and CICRYs(CICRYla,CICRYlb and C1CRY2),the interaction of C1RVE8 and CICOLs(C1COL1,C1COL2,C1COL4 and C1COL5)and the interaction of CICRYs and CICOLs.The results showed that C1RVE8 directly interacted with CICRYs and CICOLs,while CICRYs could not interact with CICOLs.Those results indicated that C1RVE8 might be a signal protein involved in CICRYs-mediating SD signal to regulate the floral transition of C.lavandulifolium.In conclusion,the author illustrated a model depicting C1CRYs-mediating photoperiod signal to regulate the floral transition of C.lavandulifolium.The CICRYs in leaves could response to the SD signal.On the one hand,CICRYs act to upregulate the circadian clock related genes,CICOLs and C1FT1,leading to the floral transition of C.lavandulifolium.On the other hand,C1RVE8 might be involved in the signal transduction pathway of CICRYs.The present study laid a foundation for effectively promoting the sensitivity of chrysanthemums to SD conditions.