Map-based Cloning And Functional Analysis Of DFO1 Gene Involved In Regulating Floral Organ Identity In Rice

The grass family(Poaceae)contains many important crops,including rice(Oryza sativa),wheat(Triticum aestivum),barley(Hordeum vulgare)and maize(Zea mays)and so on.Among them,rice as an important crop has been selected as the model plant for functional genomics studies of crops,and its spikelet morphogenesis is crucial for cereal yield.The "ABC" model based on studies from dicot species,such as Arabidopsis thaliana and Antirrhinum majus,has been widely accepted.In monocots,the study of flower development is relatively backward compared with the dicots.Recently,according to the studies of rice floral organ mutants,the mechanism of rice floral development is gradually clear,and also established an "ABCDE" model that applicable to rice floral development.Studies have shown that the two lodicules of the rice flower is homolog of the dicot petals,while the lemma or the palea,which is equivalent to the dicot sepal as the first whorl remains elusive.In addition,several epigenetic repressors that are involved in the regulation of floral organ morphology have characterized in Arabidopsis thaliana.However,other mechanism that regulating rice floral organ development has not been well explored,except the MADS-box genes are involved in the regulation of rice flower development.Therefore,further studies will be needed to identify more genes that involved in floral organ development to elucidate the regulation mechanism of rice flower development.While the identification of rice floral organ development related to the lemma and palea has important theoretical significance for their identities.In this study,we systematacially characterized an abnormal floral organ recessive mutant dfol and analyzed the molecular mechanism for defects of floral organs.The main results are as follows:(1)Two mutants with the similar phenotypes of floral morphology were isolated from an M2 population of 60Co-irradiated variety 93-11(Oryza sativa,indica).The mutants resembled wild-type in the vegetative stage,but it took slightly longer to flower and its height was significantly reduced.In addition to plant architecture,another obvious defect was the morphology of the spikelet.The palea of the mutant spikelet was much smaller and incurved to the floral central;the lodicules partially lost its identity and some took the identity of pistil;the number of stamens per floret was reduced and some stamens resembled the pistil-like organs;the pistil were ecotopically grown in the mutant and the number was increased.Accorrding to the phenotypic defects of the floral morphology,we named the mutant as deformed floral organ1.When crossed with 93-11,the phenotypes of F1 is like the wild-type,and the ratio of normal to abnormal plants in F2 population is nearly to 3:1,suggesting that the mutant phenotype is controlled by a single recessive gene.SEM analysis at the early floral organ stage suggested that the identities of floral organ were altered in early floral development of dfo1-1 mutant.In addition,SEM analysis revealed that the mrp of the wild-type palea had a smooth surface,whereas the identity of the margin of rice palea(mrp)in the dfol-1 palea was barely established and had a rough surface like that of the wild-type ovary.Meanwhile,some stigmatic papillae-like tissues were observed on the tip of the mrp in dfol-1 mutant.Through these phenotypic observations,we hold that homologous transformation and ectopic growth of the floral organs occurs in dfol mutant.(2)We isolated the mutant gene by map-based cloning.1058 recessive plants showing the dfol phenotypes were chosen from the F2 progeny of a cross between dfol and 02428,and the mutated locus was further mapped to a 60 kb region.And fourteen ORFs were predicted by the Rice-GAAS in this region(rice genome automated annotation system).Sequence analysis showed that the second ORF differed between wild-type and both mutants,including single base change or deletion and causing a premature stop.Functional analysis found that this ORF encodes a putative ortholog of Arabidopsis EMBRYONIC FLOWER1(OsEMF1).Complemention analysis demonstrated that the OsEMF1 is the DFO1 gene.(3)Real-time PCR analysis indicated that DFO1 was constitutively expressed in all organs,including young roots,stems,leaves and panicles.Further,GUS staining of the pDFO1::GUS transgenic plants suggested that DFO1 was expressed in all organs.Homology analysis found that the DFOl is orthologous to Arabidopsis thaliana EMBRYONIC FLOWER1(EMF1),but the similarity is low,only have three conserved regions.Several domains were identified in the DFO1 polypeptide,including four potential nuclear localization signals(NLS),a putative ATP/GTP binding motif(P-loop)and an LXXLL motif.Subcellular localization of the DFO1 protein showed that the DFO1-GFP fusion protein was localized in the nucleus,and the amino acids 328-410 of DFO1 are necessary for its nuclear localization.Yeast two-hybrid assay suggested that the LXXLL motif of C terminal(Ct)fragment can repress the transcriptional activity.These results suggested that DFO1 encodes a nuclear protein.(4)Expression analysis the expression of floral organ identity genes in dfo1-1 found that the expression of C-class genes was increased,and RNA in situ hybridization suggested the OsMADS58 was ecotopically expressed in the palea and lemma of dfo1-1.Overexpression of OsMADS58 resulted in phenotypes similar to those of dfol mutants,suggesting DFOl might negatively regulate the expression of OsMADS58.(5)The yeast two-hybrid,pull-down(in vitro)and BiFC assays verified that DFO1 could interact with OsMSI1 and OsiEZl,and we hypothesized that DFO1 participates in PcG-mediated gene repression by the H3K27me3 modification.Chip-real-time PCR assays found that the H3K27me3 modification of OsMADS58 was dramatically decreased.Therefore,DFO1 appears to mediate the expression of OsMADS58 through epigenetic regulation.