Molecular Mechanism Of Protein Kinase PMF1 Regulating Heading Date In Rice

Heading date is an important agronomic trait,which determines the regional distribution and yield of rice varieties.Therefore,it is of great value and practical significance for crop genetic improvement to isolate and clone the regulatory flowering genes in rice.In this study,we found that phosphorylated Os FD1 to form a florigen activation complex(FAC)through 14-3-3 binding with long day florigen RFT1.In addition,we characterized a CIPK protein kinase,the phosphorylation modifier of Os FD1(PMF1),which interacts with and phosphorylates Os FD1.PMF1 influences the expression of Os MADS14/15/18/34 genes and regulates the floral transition in rice under long-day conditions.The main findings were described as follows: 1.Nuclear accumulation of the RFT1/Gf14c/Os FD1 complex requires phosphorylated Os FD1.Both in vitro pull-down assays and in vivo bimolecular fluorescence complementation assays(Bi FC)confirmed that long-day florigen RFT1 interacts with the 14-3-3 protein.In vitro phosphatase treatment and pull-down assays showed that 14-3-3 protein(Gf14c)interacts with phosphorylated Os FD1,does not dephosphorylated Os FD1.In addition,in vivo bimolecular fluorescence complementation and subcellular localization assays showed that Os FD1 protein interacted with RFT1/Gf14 c complex to form FAC,which locates in the nucleus.2.Os FD1 promotes flowering in rice.The crispr-osfd1 mutant were generated by using CRISPR/cas9 system.Compared with the control Zhonghua11(ZH11),the heading date of the crispr-osfd1 lines was delayed by 17 days under natural long-day conditions in Wuhan and by 9 days under short day conditions.In addition,we generated the Os FD1 overexpressing plants Os FD1-OE and Os FD1(S192A)overexpressing plants Os FD1(S192A)-OE.Under long-day conditions,the Os FD1-OE plants heading 8 d earlier than control Zhonghua 11(zh11),while the Os FD1(S192A)-OE plants showed similar heading date as wild type plants.It is speculated that Os FD1 needs phosphorylated S192 residues to promote flowering in rice.3.PMF1 physically interacts with Os FD1 in the nucleus.The yeast two-hybrid assay identified that protein kinase PMF1(LOC?07g48760)is a Os FD1 interacting protein.The N-terminus(aa 1-313)of PMF1 interacts directly with the C-terminus(aa 124–195)of Os FD1.Protein interaction between PMF1 and Os FD1 was further verified by a pull-down assay and Bi FC.Subcellular localization experiments showed that PMF1 and Os FD1 colocalized in the nucleus.Bi FC also showed the CFP fluorescence of PMF1-Os FD1 interaction in the nucleus of rice protoplasts.Thus,PMF1 interacts with Os FD1 in the nucleus.4.PMF1 modulates the heading date under long day conditions,also affects the spikelets number and branches number per panicle.The Tos17 insertion mutant pmf1 of PMF1 gene was identified.In addition,the heading date of pmf1 plants was delayed by 20 days compared with that of wild-type plant under LDs,but consistented with that of ZH11 under the SDs.Regarding other agronomic traits,pmf1 exhibited an increased panicle size(more panicle branches and spikelets)compared with that of wild-type plant under LDs.Genetic complementation testing confirmed that PMF1 gene resumed the late heading phenotype of pmf1 mutant.5.Protein kinase PMF1 phosphorylates Os FD1.In vitro kinase assays showed that PMF1 protein has kinase activity and can be autophosphorylated.PMF1 phosphorylates the serine residue at position 192 of Os FD1 protein(S192).In addition,immunoblot analysis showed that the amounts of phosphorylated Os FD1 in pmf1 plants was significantly lower than that in ZH11 under long day conditions.These results revealed that PMF1 is likely responsible for phosphorylation of Os FD1 under long day conditions.6.PMF1 regulates the expression of Os MADS14/15/18/34 genes via FAC.We examined the expression levels of Os MADS14/15/18/34 in the shoot apex of pmf1 and crispr-osfd1 plants under LDs and SDs,respectively.Compared with ZH11,Os MADS14/15/18/34 expression levels were significantly reduced in pmf1 and crispr-osfd1 under long-day conditions,but no significant change under short-day conditions.In addition,Os MADS14/15/18/34 abundance was markedly increased in Os FD1-OE but remained nearly the same in Os FD1(S192A)-OE compared with ZH11 plants.Thus,PMF1 regulates the expression of Os MADS14/15/18/34 genes under long-day conditions.7.PMF1 affects the expression of Ghd7 and Hd1 genes under long day conditions.PMF1 shows diurnal expression patterns with a trough in the light under both SDs and LDs,but its abundance is higher under LDs than that under SDs.In addition,the flowering repressors Ghd7 and Hd1 expression were increased under LDs,and the flowering activators Ehd1 and RFT1 exprssion were suppressed in pmf1 under long-day conditions.However,yeast two-hybrid assays showed that PMF1 does not interact with Ghd7,Hd1 and Ehd1.Thus,PMF1 may affect rice flowering under long-day conditions through Ghd7 and Hd1 pathway.In summary,we revealed the molecular model of FAC formation with florigen RFT1.RFT1 interacts with Gf14 c in the cytoplasm,resulting in the RFT1/Gf14 c complex entering the nucleus and binding to phosphorylated Os FD1 to form a ternary protein complex.Moreover,we identified a protein kinase gene PMF1,pmf1 mutant delayed heading under long day conditions and normal heading under short day conditions.PMF1 interacts with and phosphorylates Os FD1.Therefore,we propose that PMF1 phosphorylates Os FD1,which is required for nuclear accumulation of the FAC and initiates reproduction during long-day flowering in rice.