The Mechanistic Analysis Of CIB3-Regulated Floral Transition In Arabidopsis Thaliana

The basic/helix-loop-helix(bHLH)proteins belong to a superfamily of transcription factors that exists widely in eukaryotes.bHLH proteins consist of two functionally distinct regions.The basic region at the N-terminal which contains several conserved basic amino acid residues is involved in DNA binding.The core DNA sequence recognized by the basic region is E-box or G-box.And the HLH region at the C-terminal mainly consists of hydrophobic residues and functions as a dimerization domain.The bHLH transcription factors play important roles in plant growth and stress response.CIB3(cryptochrome-interacting basic-helix-loop-helix),located in the bHLH subfamily 18,is more closely related to CIB1(cryptochrome-interacting basic-helix-loop-helix)which acts as a key component in cryptochrome 2(CRY2)mediating blue light signal transduction pathway.In this study,the function and the mechanism of CIB3 in regulating photoperiodic flowering were analyzed using transgenic and biochemical approaches,which is of great significance to understand the elaborate labor-division of different CIB members in the regulation of floral transition.The main results of this work are as follows:1.The expression of CIB3 gene in young leaves,rosette leaves,flower buds and fully open flowers.The putative CIB3 protein contains the basic region at the N-terminal and the HLH domain at the C-terminal.The nuclear location sequence(NLS)was found in theamino acid sequence of CIB3 protein.CIB3 gene,being predicted to have two splice variants,was mainly expressed in the form of first splice variant in the young leaves,rosette leaves,flower buds and fully open flowers.CIB3 was highly expressed in the young leaves and open flowers.Multiple E-box elements present in the promoter sequence of CIB3 gene.The expression of CIB3 was up-regulated by light,whereas inhibited by darkness.Lost-of-function mutation of RDR6,a gene encoding an enzyme for inducing transgene silencing,does not affect the alternative splicing and the expression pattern of CIB3.2.CIB3 promotes photoperiodic flowering.The genome sequence of CIB3 was amplified by PCR and inserted into the vector pEGAD,driven by its native promoter.The resulting construct was introduced into rdr6 mutant through floral dipping method.The transgenic lines of CIB3/rdr6 were confirmed by Western blot assays.Under long day conditions,CIB3/rdr6 plants flowered significantly earlier than rdr6 plants.The real-time quantitative PCR(q PCR)analysis showed that the transcript levels of GI,FT,CCA1 and LHY in the CIB3/rdr6 plants are higher than those in the rdr6,indicating that CIB3 promotes flowering through photoperiod pathway.3.CIB3 acts dependently on other CIB rather than CRY2.The bimolecular fluorescence complementation(Bi FC)assays confirmed that CIB3 interacted with CIB1,but not with CRY2.CIB3/cry2 plants flowered significantly earlier than cry2 plants,whereas CIB3/cib125 plants showed similar flowering phenotype as cib125 under long day conditions.These results showed that the ability of CIB3 to promote flowering depends on at least one of CIB1,CIB2 and CIB5,but not on CRY2.4.CIB3 regulates photoperiodic flowering through promoting the transcription of GI.The results from dual luciferase assays showed that CIB3 could bind to the promoter of GIGANTEA(GI)gene,and CIB1 and CIB3 synergistically function in promoting the activity of GI promoter in plant cells.The DNA Pull down assays confirmed that both CIB1 and CIB3 bind to the GI promoter in HEK293 T cells.ChIP-q PCR showed that CIB3 could bind to GI promoter in vivo.CIB3/gi-1 plants showed similar flowering phenotype as gi-1 under long day conditions,indicating that the function of CIB3 in promoting flowering depends on GI.5.The ability of CIB3 to promote flowering depends on FKF1,which promotes the interaction between CIB3 and CIB1.CIB3/fkf1 plants flowered at the same time as fkf1 under long day conditions,confirming that CIB3 acts dependently of FLAVIN-BINDING,KELCH REPEAT,F-BOX(FKF1)protein.The split luciferase complementation(Split-LUC)assays confirmed that FKF1 enhanced the interaction of CIB3 with CIB1 responding to blue light in HEK293 T cells.ChIP-q PCR showed that CIB3 regulates the transcription of GI gene dependent on FKF1 in vivo.