Alternative Expression Of RcCOL4 In Short Day And RcCO In Long Day Facilitates Day-neutral Response In Rosa Chinensis

Rosa hybrida belonging to a woody plant of the genus Rosaceae is one of ten traditional flowers in China and the four major cut flowers of the world.It is popularized worldwide with good reputation of ’Queen of Flowers’,and has high ornamental and economic value because of its fragrance,abundant colors,architecture and continuous flowering.Continuous flowering refers to plant flowering at any favoralble growth contiditions regarless day-length,hence,is generally considered as day-neutral response.In fact,there are three different flowering modes in rose plants:once-flowering(OF),continuous flowering(CF)and occasionally re-blooming(OB)genotypes.So,rose plants are regarded as perfect materials to study the mechanism of flowering regulation in woody plants.Up to present,there are limited studies on the regulation mechanisms of CF domestic and overseas.Therefore,it is particularly significant to study the underlying mechanism of CF with the perennial woody roses for expanding our knowledge about plant floral formation.The BBX family genes are a functionally diverse class of zinc finger transcription factors encoded by a highly conserved group of genes across multicellular species.They are characterized by the presence of one or two B-box motifs in the N-terminal and recognized to play an important role in plant photoperiodic regulation and photomorphogenesis.Therefore,it is feasible and significant to explore the mechanism of BBX family genes in the regulation of flowering time of continuous flowering roses.In the present paper,we performed physiological experiments,bioinformatics analysis and genetics verification,initially revealed two functional BBX family genes in the regulation of continuous flowering and the molecular mechanism of them.1.To confirm whether CF roses are DN plants,we compared the flowering time of’Old Blush’ cuttings transplanted in long day(LD)and short day(SD)conditions.As a result,cutting seedlings bloomed at 43 d under LD in contrast to 44 d under SD condition.The difference of flowering time between SD and LD was not statistically significant,therefore,supporting the DN response of rose plants.2.To clone BBX family genes in rose,we screened the new published genome database of Rosa chinensis ’Old Blush’and identified eighteen non-redundant BBX genes that contained one or two BBX domains.The number of BBX genes in ’Old Blush’ is about half of that in Arabidopsis and distribute in all five groups.Group I subfamily BBX genes are recognized to play a prominent function in flowering regulation and photoperiod response.In ’Old Blush’,three genes were classified into the structure group I subfamily.Accordingly,the genes were designated as RcCO(RcBBX1),RcCOL4(RcBBX5)and RcCOL5(RcBBX6)following the nomenclature system.Subsequently,the expression levels of RcCO and its closest family members RcCOL4 and RcCOL5 were examined every 4 h in a 24-h cycle starting at the onset of light.The results clearly showed circadian regulation of the genes categorizing them according to the expression differences between SD and LD condition:higher in LD and lower in SD(RcCO),lower in LD and higher in SD(RcCOL4),lower or higher alternately(RcCOL5)in SD and LD.Collectively,these results suggested their potential roles in photoperiod-dependent flowering time.3.To gain the genetic evidence of the biological functions of RcCO,RcCOL4 and RcCOL5,VIGS(Virus-induced gene silencing)technique was employed to silence the genes in Rosa chinensis ’Old Blush’,followed by recoding their respective flower phenotypes.Specifically,flowering of RcCO-silenced plants delayed 14 d in LD and 9 d in SD,while flowering of RcCOL4-silenced plants delayed 4 d in LD and 7 d in SD,whereas flowering of RcCOL5-silenced plants delayed 6 d in LD and SD.These results clearly indicated that RcCO,RcCOL4 and RcCOL5 were all flowering activators in rose plants and essential for normal flowering under SD and LD conditions.It was noteworthy that only silencing of either RcCO or RcCOL4 induced the difference of flowering time between SD and LD,thus,disturbed the DN response of rose plants,ruling out the function of RcCOL5 in DN response.Furthermore,the identical flowering time between RcCO/RcCOL4 double-silenced plants and RcCO-silenced single mutants implied RcCOL4 and RcCO are under the same regulatory pathway.4.To further test the functions of RcCO and RcCOL4 in Arabidopsis,we over-expressed them in Col wild type(WT)as well as co mutant background.Based on the rosette leaf number under LD condition,the flowering time of over-expression lines were accelerated significantly in WT backgrounds.In terms of the co mutant,the flowering time was delayed significantly to 18 rosette leaves in comparison with 10 in WT,over-expression of RcCO decreased the number of rosette leaves to 9,while no obvious phenotype was observed upon over-expression of RcCOL4 in co mutant background.Furthermore,AtCO most likely acted downstream of RcCOL4 and was essential for its function in Arabidopsis,consistent with the previous result in roses.5.CO/FT model is conserved in model plants.To confirm this model in rose,we cloned the promoter of RcFT,and identified RcCO binds to CORE motif(CCACA)in the promoter of RcFT by transient Agrobacterium tumefaciens infiltration assays,yeast one-hybrid and EMSA.We also confirmed that RcCOL4 certainly enhanced the binding activity.To further understand the relationship between RcCO and RcCOL4,we identified the interaction between them by split luciferase complementation assays,yeast two-hybrid and Pull-down assays.Then,we also confirmed BOX1 in the RcCOL4 is essential for the interaction between RcCO and the the function of RcCOL4 in RcFT promotion.