Function Characterization And Mechanism Dissection Of StPHYF Related To Tuberization In Potato

Potato is the fourth most important food crop,ensuring food security particularly in developing countries because of its high yield,adaptation to diverse environmental conditions and high nutritional values.However,the sensitivity of tuberization to photoperiod limits use of new varieties which are not adaptable to long days.Therefore,exploring to the mechanism underlying photoperiodic tuberization in potato has applicable and theoretical importance.Lots of genes have been reported recently to be involved in potato tuberization mediated by photoperiod.In long day(LD)conditions(16h light/8 h dark),protein StCOL1 activates the expression of StSP5 G via binding the promoter of StSP5 G to repress the expression of StSP6 A,preventing the stolon tips from tuber initiation.In LD,StPHYB suppresses tuberization through stabilizing the StCOL1.However,information gained so far is inadequate for drawing a conclusion how potato tuberization is regulated by photoperiod.Based on our previous research that silencing by RNAi of StPHYB and StPHYF induced microtuber formation in LD,whereas the other three potato phytochromes(StPHYA,StPHYB2,StPHYE)showed no effects on LD-tuberization.The present research focused on dissecting how phytochromes regulate potato photoperiodic tuberization.The main results are as follows:1.The transgenic lines(2-3 for each of the five potato phytochrome genes)were further analyzed for tuberization in vitro under LD conditions.We confirmed that silencing StPHYF and StPHYB significantly induced microtuber formation,while silencing StPHYA,StPHYB2 and StPHYE had no obvious impacts,revealing that StPHYF and StPHYB play inhibitory roles in potato microtuberization in LD.There were no effects of silencing StPHYF and StPHYB on the expression of other phytochrome genes observed.To further confirm the function of StPHYF in potato tuberization,an StPHYF-RNAi construct was transformed into potato E109 plants,a strict short day(SD)genotype that can only form tubers in vitro and in vivo under short days.The results demonstrated that under LD conditions,suppressing StPHYF led to normal tuber formation in both tissue-culture and pot-grown plants,whereas the wild-type control showed no tubers in the same conditions.It was also reinforced that silencing StPHYF had no impacts on the expression of the other phytochrome genes under diurnal conditions.Our results first evidenced that StPHYF plays important roles in inhibiting tuberization in LD.2.Because of a similar function of StPHYF and StPHYB in photoperiodic tuberization,we speculated that they might be involved in the same pathway to modulate potato tuber initiation in response to day length.However,it remains to be determined whether StPHYF and StPHYB act independently or work together to exert a synergistic function in control of potato tuber initiation.Through the experiments of Y2 H,BiFC and CO-IP,the results revealed that StPHYF interacts with StPHYB to yield a heterodimer.Moreover,we found that the heterodimer is possibly formed via the binding of the C-terminus of StPHYF and the N-terminus of StPHYB.3.It was reported that StPHYB stabilize the StCOL1 protein.To investigate the role of StPHYF in this pathway,the abundance of the StCOL1 protein was monitored in the StPHYF-silenced E109 line taking advantage of the specific StCOL1 polyclonal antibodies.During the 12 h light period,while untransformed control plants showed an increased or relatively stable level of StCOL1,silencing of the StPHYF gene resulted in a prominent reduction of StCOL1 protein abundance.The effects of suppressing StPHYB on StCOL1 were also tested in StPHYB-silenced E3 plants.Similar results were obtained,showing a much lower StCOL1 protein content in the transgenic line compared with control.These results revealed that StPHYF plays a central role in stabilizing the CONSTANS protein StCOL1,and this stabilization might be achieved by interacting with StPHYB.4.To dissect the effects of StPHYF on signal transduction in the CO-FT regulon,we assessed the expression of genes related to tuberization(StCOL1,StSP5 G,StSP6A and StCDF1).The results showed that,under LD conditions in vitro,compared with the expression observed in untransformed control plants,the StSP5 G gene was highly repressed,whereas the StSP6 A gene was significantly induced,but little effects on StCDF1 and StCOL1.Taken together,these findings suggest that StPHYF,possibly acting in cooperation with StPHYB,plays roles in the CO-FT regulon by upregulating StSP5 G and repressing StSP6 A in leaves.The expression of StCOL1,StSP5 G,StSP6A and StCDF1 in the leaves of the transgenic lines of StPHYFi-E109 cultivated in growth room was similar with the results in vitro.However,to our interesting,the expression of StCDF1 and StSP6 A was induced in the stolon of the transgenic lines of StPHYFi-E109,while the expression of StSP5 G was not altered in stolons,compared with the control.It is speculated that there may be another factor which can transport to stolons together with StSP6 A to control the expression of StCDF1.5.Suppression of StPHYF may modulate signal transmission from leaves to stolons for tuber initiation under long days.To test this hypothesis,apex scions of the StPHYF-silenced E109 transgenic line grown in LD conditions were grafted onto wild-type E109 stock.All the stocks grafted with StPHYF-silenced scions produced tubers under LD conditions,whereas the self-grafted control did not form any tubers,strongly suggesting that the signal induced by suppressing StPHYF in leaves is transmitted to underground to stimulate tuber formation at the stolon tips.To detect the transduction of this signal,the expression of StSP5 G,StSP6A and StCDF1 was analysed in scion leaves and stock stolons.The expression of StSP6 A and StCDF1 was dramatically upregulated in the stolon of the StPHYF-silenced scion plants compared with the wild-type scion plants.Therefore,there actually be another factor which can transport to stolons together with StSP6 A to control the expression of StCDF1.The accumulation of the transcription factor StCDF1 in stolons of StPHYF-silenced lines might further enhance the accumulation of StSP6 A transcripts in the place,which would reinforce the involvement of StPHYF in the pathway of tuberization mediated by photoperiod.6.We screened the proteins potentially interacted with StPHYF and StPHYB,for that the Yeast Two-Hybrid Library was constructed with potato genotypes(E20,E26 and E108)distict in photoperiodic tuberization charaterastics.Totally 79 potential target proteins were selected,among them bHLH,Zinc finger,NAC transcription factors and two kinases were found specificly interacted with StPHYF,and the later may phosphorylate phytochromes or proteins related to phytochromes.Simultaneously,we performed the RNA-Seq of the transgenic lines of E109(PHYFi-E109-13,PHYFi-E109-14)and E3(PHYFi-E3-9 and PHYFi-E3-15)in comparison with the wild type corresponding control grown in LD aiming to identify the genes regulated by StPHYF.The results yielded 32 genes that may be downstream and modulated by StPHYF,of which the MADS box transcription factors could be the potentials associated with tuber formation,and terpen biothynthesis genes may be related to the secondary metabolitese for long-distance signaling in tuberization.These results may open a new avenue and strategy for further research.