Molecular Mechanism Of FPF1s In Flowering Regulation In Brachypodium

Flowering sets the transition from the vegetative to the reproductive development of plants.To maximize reproductive success,plants integrate numerous environmental cues and endogenous signals,and generate a complex and sophisticated regulatory network to initiate flowering on time.The Study on the genetic mechanism in flowering time regulation would provide great insights into crop heading-date breeding for yield improvement and regional adaptability.FLOWERING PROMOTING FACTOR1(FPF1)has been shown with conserved functions in promoting flowering in many plants,including Arabidopsis,rice,and cotton;however,the underlying molecular mechanism remains unknown over the past 20 years.Brachypodium distachyon,the ideal model plant of temperate grass and cereals like wheat and barley,was used in this study to investigate FPF1 roles in flowering control.The biological function and molecular machinery of two FPF1-like proteins in Brachypodium,FPF1 and FPF7,were characterized,and the main conclusions are summarized as follows:(1)qRT-PCR results showed that FPF1 and FPF7 were specifically induced by long-day conditions(LDs)and exhibited significantly higher expression than the other orthologs.(2)Plants over-expressing FPF1 or FPF7 displayed a clear delay in flowering,while their single and double mutants exhibited early-flowering phenotypes,suggesting the negative roles of FPF1 and FPF7 in flowering initiation under LDs.(3)LCI,BiFC,and Co-IP results showed that FPF1 and FPF7 interacted with FLOWERING LOCUS T 1(FT1)protein,the florigen in Brachypodium,and also the other two components of the florigen activation complex(FAC),FD1 and Gf14 b.(4)The further protein-protein interaction assays,EMSA,and Luciferase assays indicated that the interactions between FPF1 and FPF7 with FAC components were both detrimental to FAC assembly and interrupted FAC binding to DNA elements,thereby repressing the expression of FAC downstream gene,VERNALIZATION 1(VRN1).(5)The temporal and spatial expression results showed that FPF1 and FPF7 were mainly expressed in plant leaves at the early vegetative stage.Further qRT-PCR,RNA-seq,and genetic analysis indiacated that through repressing VRN1 expression in leaves,FPF1 and FPF7 impaired the positive feedback loop between FT1 and VRN1,which subsequently led to the restriction of florigen production,hence avoiding premature flowering at the juvenile stage.(6)EMSA,ChIP-PCR,Luciferase assays,and qRT-PCR results showed that at the late vegetative stage,VRN1 directly associated with the FPF1 promoter and repressed its transcription,thereby releasing itself from the inhibition of FPF1 protein,which further promoted FT1 expression in leaves,resulting in sufficient florigen for transport to the shoot apical meristem(SAM)to timely trigger flowering.Overall,two FPF1-like genes are found,for the first time,that function as flowering repressors in our study,in contrast to the previously reported functions of FPF1 family in other species.On this basis,we further elucidate when,where,and how FPF1 and FPF7 negatively regulate flowering in Brachypodium.Our results might help develop the understanding of plant flowering regulatory networks,and provide a theoretical reference for Triticeae crops heading-date breeding and genetic improvement.