The Molecular Genetical And Biochemical Study And Function Investigation Of Photo-responsive F-box Protein FOF2 During Floral Initiation In Arabidopsis

Flowering is the turning point in the phase transit io n from vegetat ive to reproductive growth.The flowering t ime direct ly affects seed yield and successful propagat ion in plants.The control of flowering init iat ion t ime is extremely complex,external environmental factors and internal genet ic factors affect mult iple signals to precisely control the flo wering t ime.A series of flo wering regulatory genes reported in Arabidopsis thaliana,and there are six signaling pathway that control the flowering time in plants.However,the mo lecular mechanisms of the mo lecular cross-talk between signaling pathwaysare not ful ly understood.In this study,by screening the Arabidopsis F-box gene excessive expressio n mutant library,we ident ified an F-box gene that involved in Arabidopsis flowering and named FOF2(F-box of flowering 2).We successfully obtained FOF2 overexpressed plants,dominant negat ive mutants and fof2fol1 double mutants.We estimated the role of FOF2 in regulat ing the flowering in Arabidopsis by comparing the expressio n of key flowering genes in transgenic plants and wild t ype,analyzed the physio logical and biochemical characterist ics of FOF2,and determined the locat io n of the FOF2 in the network by comparing the expression differences between the flowering mutant and the wild t ype.In this study,we researchedthe funct ion of FOF2 in flowering regulat ion onupper aspects.Results as follows:(1)FOF2 belo ngs to the B4 subfamily of the Arabidopsis F-box superfamily.The phylogenet ic tree analysis of B4 subfamily members showed that FOL1(FOF2 like 1),FOL2,FOL3 and FOL4 had the closest relat ionship wit h FOF2 evo lut ion.FOF2 contains an F-box domain at the N-terminus,an LRR domaininmediate,and an FBD domain at the C-terminus.The overall amino acid sequence alignment showed that the sequence ident it y between FOF2 and FOL1 was 87%,wheras that of FOF2 and FOL3 was about 46%.(2)Overexpressio n of FOF2 resulted in late flowering phenot ype under long day and short day condit ions in Arabidopsis thaliana,but FOF2 delet ion did not affect the flowering t ime.However the dominant negat ive mutant Myc FOF2?F and the double mutant fof2fol1 of FOF2 and its homo logous gene FOL1 constructed by CRISPR / Cas9 system show early flowering phenot ype under both long day and short day condit ions,suggest ing FOF2 is a flowering suppressor,with FOL1 funct ional redundancy.(3)FOF2 expressed in different t issues and organs,among which the expressio n level of FOF2 in root,stem leaves and fruit folders was higher,and the expressio n level of FOF2 was relat ively low in seedling,indicat ing that FOF2 was const itut ive expressio n.FOF2 transcript ion and protein expressio n levels decreased during floral transit io n,which is consistent with its negat ive regulatory flowering funct ion.Transient ly transformed tobacco expressing GFP-FOF2 or FOF2-GFP fusio n proteins were analyzed,a GFP signal was detected in the nuclei of epidermal cells,suggest ing that FOF2 was localized in the nucleus.(4)Bi FC and Co-IP results showed that FOF2 interact with ASK14,but could not when lost the F-box domain,suggest ing that F-box is required for FOF2 to funct ion properly in SCF complex formation.(5)By comparing the expressio n of flowering related genes in wild t ype,transgenic plant and delet ion mutant,we found that the expression of flowering suppressor gene FLC increased in FOF2 overexpressing plants and decreased in mutants.Genet ic analysis revealed that the lack of funct ional FLC gene could completely inhibit the late flowering phenotype of FOF2 overexpressing plants,indicat ing that FOF2 inhibited flowering by promoting FLC expression.(6)Different photoperiod and GA treat ment had no effect on the late flowering phenot ype of FOF2 overexpressing plants,and vernalizat ion could rescue the late flowering phenot ype of FOF2 overexpressing plants,which indicated that FOF2 may affect plant flowering through the autonomous pathway.By comparing the expressio n of FOF2 in various flowering-t ime mutants and corresponding wild-t ype,we found that FCA could inhibit the expressio n of FOF2,suggest ing that FCA may be located upstream of FOF2.(7)FOF2 have light sensit ivit y,light induce s the m RNA expressio n of FOF2,FOF2 protein was accumu lated in light and degraded by the 26 S proteasome pathway in darkness.The plant photomorphogenesis inhibitor COP1 did not affect the stabilit y of FOF2,and FOF2 had no effect on the elongat ion of hypocot yls in Arabidopsis under different light condit ions.In short,light regulate the plant flowering by controlling the expressio n of FOF2.