The Molecular Mechanism Of Flowering Time Regulation By RcPIFs Under Shade In Rosa Chinensis

Light is not only a source of energy for plants,but also one of the important environmental signals,which plays a vital role in the growth and development of plants.Light participates in every stage of plant growth and development:from seed germination,photomorphogenesis,flowering to fruit development.Rose（Rosa hybrida）,as one of the top ten traditional famous flowers in China,is known as the‘Queen of Flowers’and has an extremely high commercial value and unique cultural connotations.It has the ability to continuously bloom multiple times in a year under suitable environmental conditions.This continuous flowering habit of the rose makes it an excellent material for studying the mechanism of flowering regulation,however,insufficient availability of light will seriously affect the quantity and quality of rose blooms.Up to present,there are limited studies on the mechanism of light regulating flowering in woody plant.Therefore,it is particularly significant to study the underlying mechanism of light intensity on flower regulation of woody perennial rose and to screen it for shade or low-light tolerance rose varieties.PIFs are the transcription factors with basic helix-loop-helix（b HLH）domain.PIF family genes are commonly exist in plants and play an important role in photomorphogenesis and light signal transduction.Therefore,the expression pattern of PIF family genes under different light intensities is a good implication to explore its functional mechanism in rose flowering.In this study,the ancient Rosa chinensis‘Old Blush’,whose genome had been sequenced,was used as a study material.During this study,a series of molecular,biochemical,and genetic analysis the function of the three PIF family genes in rose flowering in response to light intensity.1.To clarify the response pattern of rose flowering to shading conditions,the cutting seedlings of Rosa chinensis cv.‘Old blush’with the same growth state were transferred to two contrasting light intensity regimes including LL(92μmol·m^-2·s^-1-W)and HL(278μmol·m^-2·s^-1-W)with or without a simulated shade（EOD-FR）treatment,and the number of days to flowering were counted.The results showed that the flowering time of‘Old blush’under high light was 47 days and that of low light was 66 days,indicating that low light delayed the flowering time of rose.Although the expression of flowering genes was consistent with the flowering time,the addition of far-red light under different light intensities had no significant effect on the flowering time.2.In order to study the function of PIF genes in rose,we screened the newly published genome database of Rosa chinensis and identified seven putative non-redundant PIF genes,far fewer than existed in Arabidopsis（15）.To further explore the response of PIF genes to different light intensities,the rose PIF genes Rc PIF1,Rc PIF3 and Rc PIF4 were selected for further functional characterization.Using the c DNA of the‘Old Blush’grown under different light conditions as template,the q RT-PCR was performed and the results clearly displayed that the transcription levels of the three RcPIFs were increased with the decrease of light intensity.To further test the Rc PIF protein stability in vivo,we transiently transformed each Rc PIF gene into rose seedlings,respectively.The western blotting results showed the protein degraded faster under HL than LL and dark.The results suggested RcPIFs play an important role in rose responses to different light intensities.3.To further clarify the functions of these RcPIFs,we heterologously expressed either Rc PIF1,Rc PIF3 or Rc PIF4 in Arabidopsis pifq quadruple mutant background.In contrast to the shortened hypocotyls of the pifq mutant,the dark-grown single Rc PIF-OE seedlings exhibited significantly elongated hypocotyls.However,the open apical hook and cotyledon of each Rc PIF-OE line were similar to the pifq mutant.Collectively,these data demonstrated that the RcPIFs possessed at least partially conserved functions with the PIF genes in Arabidopsis.To gain further genetic evidence of the biological functions of the RcPIFs in Rosa chinensis,VIGS was employed to silence Rc PIF1,Rc PIF3 and Rc PIF4 either separately or in combination,followed by the observation of their respective flowering phenotypes.The results showed that compared with the control line under LL conditions,the Rc PIF3silenced line and the Rc PIF4 silenced line had a significant earlier flowering time of 3 and8 days,respectively.Compared with the control line,the flowering time of the Rc PIF1/Rc PIF3/Rc PIF4 combined silenced line was significantly earlier under both LL and HL conditions.The flowering time of the three silenced line under LL was 17 days earlier,and the time of flowering was 7 days earlier under HL as compared with the control.This implied that these three PIF genes coordinately regulate flowering in rose.4.Results of the binding anaylsis using LUC as report,Ch IP-q PCR and EMSA experiments showed that the regulation of RcPIFs on Rc FT expression depends on Rc CO.Next,Bi FC,Co-IP and yeast two-hybrid assays were further performed and verified the physical interaction between the RcPIFs and Rc CO.Moreover,the interactions between Rc PIF and Rc CO were obviously affected by light intensity.Therefore,RcPIFs regulated the flowering of rose under different light intensities by affecting the stability of the RcPIFs-Rc CO complex which regulates the expression of RcFT.