Molecular Mechanism Of Rice MFT-like Genes Regulating Rice Flowering And Seed Germination

PEBP gene family is present in all eukaryote species with three subfamilies,FT-like subfamily,TFL1-like subfamily and MFT-like subfamily.In higher plants,PEBP family genes are involved in regulating plant growth and development.In various species,FT-like genes encode florigen to induce flowering and TFL1-like genes repress flowering and regulate inflorescence architecture.As the evolutionary ancestor of FT-like and TFL1-like,MFT-like genes are involved in seed dormancy and germination in many species,instead of flowering and inflorescence architecture.Rice(Orysa Sativa L.)is one of the most important food crops in the world,flowering time(also known as heading date),inflorescence architecture(also known as panicle architecture)and seed dormancy are important factors directly affecting rice yield and quality.In this study,we analyzed the function and mechanism of the two rice MFT-like genes on regulating heading date,panicle architecture and seed dormancy.We constructed the overexpression and knock-out plants of OsMFT1 and investigated their heading date and panicle architecture.OsMFT1 overexpression plants largely delayed heading date and increased number of spikelet per panicle and secondary branch.OsMFT1knock-out plants significantly decreased number of spikelet per panicle and secondary branch,and slightly accelerated heading date.Overexpressing OsMFT1 significantly suppressed the RNA level of Ehd1,Hd3a,RFT1 and OsMADS14 while Ghd7 could up-regulate OsMFT1 expression.Ghd7 RNAi plants and OsMFT1 overexpression plants were crossed to get the combination of different genotypes of Ghd7 and OsMFT1.Based on the phenotypes of the F₂plants,it is concluded that OsMFT1 acts downstream of Ghd7 in regulating heading date and panicle architecture.Thus,OsMFT1 acts upstream Ehd1 and downstream of Ghd7 in regulating flowering time.In addition,through yeast one hybrid assay,EMSA and dual luciferase transient assay,transcription factor OsLFL1 was verified to directly bind to the promoter of OsMFT1 via an RY motif and activate the expression of OsMFT1.To elucidate the mechanism of OsMFT1 regulating panicle architecture,the differential expressed genes between OsMFT1overexpression plants and wild type were examined in 0.5 mm young panicles.FZP and SEPALLATA-like genes were suppressed in OsMFT1 overexpression lines and RNA in situ verified this result.FZP and SEPALLATA-like genes are marker genes of spikelet meristem initiation during the inflorescence development.Suppression of the expression of FZP and SEPALLATA-like genes indicated that the transition from branch meristem to spikelet meristem was delayed and thus more panicle branches were produced.OsMFT2 is the other member of MFT-like subfamily in rice.OsMFT2 overexpression plants could observably delay heading date and increase the number of spikelet per panicle and secondary branch.OsMFT2 knock-out plants didn't alter heading date but significantly decreased the number of spikelet per panicle and secondary branch.To analyze the function redundancy of OsMFT1 and OsMFT2,double mutant of OsMFT1 and OsMFT2,mft1mft2,was constructed through knocking-out OsMFT1 and OsMFT2 simultaneously.Based on the phenotypes of single mutants(mft1 and mft2)and double mutant(mft1mft2),we concluded that OsMFT1 and OsMFT2 might function redundantly with other genes in regulating heading date,and these two genes had additive effect in regulating panicle architecture.In addition,mft2 and mft1mft2 were found pre-harvest sprouting.Germination test of OsMFT1 and OsMFT2 transgenic seeds revealed that freshly matured seeds of mft2 and mft1mft2 had relatively high germination rate without dormancy while mft1 and wild type not.Seeds after-ripening of OX-OsMFT1 and OX-OsMFT2 germinated significantly more slowly than wild type.Seeds without pre-harvest sprouting of mft1,mft2 and mft1mft2 were selected to perform ABA sensibility test and the results showed that the ABA sensibility of mft2 and mft1mft2 was significantly decreased compared with wild type.Further examination of RNA level of two ABA signaling marker genes,Lea3 and Rab16A,in OsMFT1 and OsMFT2 transgenic plants revealed that OsMFT1 and OsMFT2 up-regulated the expression of Lea3 and Rab16A.Through yeast two hybrid screening,3 b ZIP transcription factors(Osb ZIP23,Osb ZIP66 and Osb ZIP72)were found to interact with OsMFT1 and OsMFT2,and Bi FC assay and pull-down assay further verified the interaction.Further EMSA and dual luciferase transient assay indicated that OsMFT1 and OsMFT2probably enhanced the binding ability and activation activity of b ZIP transcription factors to the downstream genes through interacting with b ZIP transcription factors.In conclusion,rice MFT-like genes are involved in regulating heading date,panicle architecture,seed dormancy and germination.OsMFT1 has larger effect in regulating heading date and panicle architecture than OsMFT2 while OsMFT2 has major effect in regulating dormancy and germination.