The Mechanism And Biochemical Investigation Of Crosstalk Of F-box Protein FKF1 And GA Pathway During Floral Initiation In Arabidopsis

The flowering of plants is the important stage in the growth and development process.And it is an important central hub for the transformation from the stage of vegetative growth to the stage of reproductive growth in higher plants.There are various internal and external factors to affect the flowering,thus forming multiple flowering pathways.In Arabidopsis,there are approximately six signaling pathways regulating flowering time,including photoperiod,vernalization,temperature,gibberellin(GA)biosynthesis,autonomous,and aging pathways These flowering signaling pathways are independent and cross-linked and jointly control the flowering of plants at the right time.The investigation of the cross-talk between the various flowering pathways will be helpful to understand the precise regulation mechanism of plant flowering.F-box protein FKF1(FLAVINBINDING KELCH REPEAT F-BOX1)plays important roles in modulating photoperiodic flowering in Arabidopsis.Previous studies found that the fkf1 mutation strongly delays flowering under LDs.Exogenous GA₃ treatment could rescue flowering defects of fkf1 mutant.To explore how FKF1and the GA pathway work in concert in regulating flowering,we analyzed the response to GA of 35S-GFP-FKF1 and fkf1-1,fkf1-t plant to GA in flowering,and seed germination,seedling hypocotyl elongation in this study.Furthermore we analyzed the effect of FKF1 mutation on expression of GA biosynthetic and catabolic genes and the active GA₄ content.The interaction and genetic relationship of FKF1and DELLA were analyzed.Results as follows:(1)To explore how FKF1 and the GA pathway work in concert in regulating flowering,we analyzed the effect of 35S-GFP-FKF1 and fkf1 mutant to exogenous GA.The increased sensitivity of 35S-GFP-FKF1 and decreased sensitivity of fkf1mutant in flowering indicated that FKF1 positively regulated the GA response in flowering.Meanwhile,we found that the seed germination and hypocotyl elongation of fkf1 mutants were more sensitive to GA synthesis inhibitor paclobutrazol(PAC)than was wild-type.And the fkf1 mutant exhibited weaker hypocotyl elongation in response to GA than did wild-type seedlings.These results indicated that FKF1 was likely to play a positive role in regulating GA signaling.(2)To explore the mechanism of FKF1 positively regulating the GA signal,Real time quantitative polymerase chain reaction(q RT-PCR)was used to compare and analyze the expression of key genes of GA response,synthesis and metabolism in wild-type and fkf1 mutants of Arabidopsis,and results showed that the expression of GA-responsive genes(EXP8 and PRE1)were decreased in fkf1 mutants and was more sensitive to PAC.Meanwhile,expression of GA metabolic genes GA2oxs(GA2ox1,GA2ox2,GA2ox4 and GA2ox6)were significantly down-regulated in fkf1 mutant and expression of GA synthesis gene GA3ox2 was significantly up-regulated.We measured the active GA₄ content in the fkf1-1 mutant and wild-type seedlings.However,we did not find significant difference.These results suggested that FKF1could positively regulate plant response to by promoting GA signaling rather than GA synthesis.(3)To investigate whether FKF1 can affect plant flowering response to GA through DELLA protein,a key inhibitor of GA signaling pathway,western blot analysis was used to compare the levels of DELLA protein RGA in wild-type,fkf1mutant and 35S-GFP-FKF1 overexpressing plants.Results showed that the stability of endogenous RGA protein and TAP-RGA protein in fkf1 mutants was increased,while the stability in 35S-GFP-FKF1 overexpressing plants was reduced.We detected the rhythm expression of RGA protein and TAP-RGA transgenic protein under long-day conditions.And we found that FKF1 regulated the rhythm accumulation of RGA protein and promoted the degradation of RGA protein in the afternoon(light8-12 h).These results suggested that FKF1 negatively regulated DELLA protein RGA stability and rhythmical accumulation.(4)To investigate whether FKF1 directly regulates DELLA protein abundance,yeast two-hybrid assay,BIFC assay,Co-IP assay and GST pull down assay suggested that FKF1 physically interacts with DELLA proteins RGA and GAI in vitro and in vivo.And Co-IP assays using human embryo kidney 293T(HEK293T)cells both in the dark and in blue light suggested that FKF1 was likely to interact with DELLA independent of blue light.Moreover,yeast two-hybrid assay and GST pull down assay suggested that the Kelch repeat of FKF1 and the GRAS domain of DELLA were the domains for FKF1-DELLA interaction.These results suggested that FKF1 directly interested with DELLA proteins and FKF1-RGA and FKF1-GAI interactions were not modulated by blue light.(5)To investigate whether FKF1 can regulate the ubiquitination degradation of DELLA protein,the tobacco ubiquitination assay indicated that FKF1 promoted ubiquitination of RGA and GAI.Cell free assay and tobacco degradation assay suggested that FKF1 likely promoted the degradation of RGA and GAI via the 26S ubiquitin-proteasome pathway.Meanwhile the Arabidopsis system assay suggested that FKF1 likely promoted DELLA protein ubiquitination and degradation independent of blue light.These results suggested that FKF1 promotes DELLA proteins ubiquitination and degradation independent of blue light.(6)To further investigate the relationship beween FKF1 and DELLA protein in flowering,genitic analysis showed that the late-flowering phenotype of fkf1-1 was partially rescued by the rga-28 and gai-t mutations in LDs,and the triple mutant rga-28/gai-t/fkf1-1 had higher m RNA levels of FT,SOC1 and LFY,which are flowering integrators.Moreover,35S-FKF1-Myc can partially suppress the late-flowering phenotype of 35S-RGA-Flag and 35S-GAI-Flag plants under LDs and the m RNA levels of FT,SOC1 and LFY.These data suggested that FKF1 regulated flowering partially through DELLA stability.(7)To further investigate whether GA and DELLA regulated the expression of FKF1,q RT-PCR and western blot analysis suggested that FKF1 was repressed by GA at the transcriptional,but not post-transcriptional,level.And the FKF1 expression of della mutant,GAI gain-of-function mutant and RGA/GAI overexpression plants suggested that the DELLA proteins induced FKF1 expression,and GA repressed FKF1 expression in a DELLA dependent manner.These data suggested that the DELLA proteins induce FKF1 expression.