Molecular Mechanisms Of FON4(Floral Organ Number4)and ROD(Reproductive Organ Defective)in Rice Floral Organ Development

The floral meristem（FM）in angiosperms develops into floral organs whose numbers,positions,identities,and sizes lead to the diversification of flowers.The numbers of floral organs are closely associated with FM size during organ primordia initiation.Floral organ identities have been proposed to be specified by A,B,C,D,and E floral homeotic genes,as described by the‘ABCDE’model of flower development.After initiation of floral organ primordia and determination of their identities,each primordium grows to characteristic size through two consecutive phases of cell proliferation followed by cell expansion.Formation of organ primordia also creates boundaries that separate the primordia from surrounding tissues.Since cells at the boundaries usually display lower growth activity,the outermost region of the primordium forms a hollow or groove.Rice（Oryza sativa）is not only a staple food crop worldwide,but also an important model monocot plant.In recent years,many genes have been identified to be involved in rice flower developmental programs,including FM activity regulation,floral organ identity specification,organ size determination and so on.In this study,we revealed the molecular mechanisms of meristem maintenance gene FON4（FLORAL ORGAN NUMBER4,also known as FON2）and reproductive organ control gene ROD（REPRODUCTIVE ORGAN DEFECTIVE）in rice flower organ development.The main research results are summarized as follows:1.Interactions between FON4 and Floral Homeotic Genes in Regulating Rice Flower DevelopmentThe aerial parts of plants derive from the cells in the shoot apical meristem（SAM）.Maintenance of the SAM activity is dependent on the balance between differentiation and self-renewing of stem cells located in the central zone.During reproductive development,the SAM is transformed into the inflorescence meristem（IM）,which in turn produces the FM.Floral organ primordia initiate on the flanks of the FM in concentric whorls.When a fixed number of floral organs are produced,the FM ceases to have stem cell activity,and thus FM development is determinate.In the eudicot Arabidopsis thaliana,much like that in the SAM,the stem cell maintenance in the FM is based on the feedback loop consisting of the homeodomain transcription factor WUSCHEL（WUS）and the CLAVATA（CLV）ligand-receptor during early flower development.At the later stage,the termination of FM is dependent on C-class gene AGAMOUS（AG）,which is also critical for the specification of stamen and carpel identities.The AG-WUS pathway forms another feedback loop that controls the FM activity.Moreover,with respect to regulating FM development,the CLV and AG pathways seem to function at least partially independently.Inflorescence and flower development in grasses are markedly distinct from eudicot,but genetic studies have demonstrated that the CLV signaling pathway and ABCDE model are principally conserved between grasses and eudicots.The FON4 gene,an orthologue of Arabidopsis CLV3,is required for regulating floral meristem size and determinacy in rice.To reveal whether FON4 interacts with floral homeotic genes in specifying rice flower development,we constructed and analyzed the double mutants of FON4 with OsMADS15（an A-class gene）,OsMADS16（also called SUPERWOMAN1,SPW1,a B-class gene）,OsMADS3 and OsMADS58（C-class genes）,OsMADS13（a D-class gene）,and OsMADS1（an E-class gene）,respectively.We observed additive phenotype in the fon4 double mutant with the OsMADS15mutant allele dep（degenerative palea）,indicating that FON4 and OsMADS15 function in parallel pathways in specifying flower development.The effect on the organ number of the whorl 2 was enhanced in fon4 spw1,but FON4 and OsMADS16 might function independently during flower development.Double mutant combinations of fon4 with osmads3,osmads58,and osmads13 displayed enhanced defects in FM determinacy respectively,indicating that FON4 and these genes synergistically control FM activity.FON4 acts synergistically with OsMADS1 to promote the transition from spikelet meristem to floral meristem,and to maintain FM identity.The enhanced abnormality in fon4 osmads1 also suggests that there could be feedback between FM size and FM identity,and that it is difficult to maintain FM identity as FM size abnormally increases.It is possible that fon4-2 enhances the loss of stamen identity in osmads3-4 through a similar mechanism.In addition,the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant.This work also suggests that the basic framework of FM meristem regulation is conserved in the angiosperms.2.ROD encodes a C₂H₂ zinc finger transcription factor required for reproductive organ development in riceThe organ size is constant within a given species but varys among different species,suggesting that internal developmental signals determine the final size of plant organs.The duration and rate of cell proliferation and cell expansion are strictly modulated so as to maintain the intrinsic size of plant organs.The initiation of organ primordia is also associated with the formation of boundaries.Mutations in the genes that function in boundary morphogenesis cause the fusion of adjacent organs that would normally be separated.Several regulatory factors influencing organ size or boundary development have been identified in plants,but little are known about the mechanism that control the size or boundary establishment of reproductive organs in rice.Here we isolated and characterized the rod mutant in rice,which is generated by treating seeds of japonica subspecies 9522 with ⁶⁰Coγ-ray radiation.Genetic analysis revealed rod was a single recessive mutation,which displayed remarkable aberrancies in reproductive organ development.In the rod mutant,the sizes of stamens and pistils were apparently reduced,and the stamens were partially or completely fused at the filament regions.Moreover,the rod mutant displayed complete male and female sterility.Scanning electron microscopy（SEM）analysis showed that the epidermal cells of the reproductive organs of rod were apparently reduced in size as well as in number compared to those of wild type,suggesting that both cell proliferation and cell expansion are inhibited in the mutant.Paraffin section analysis indicated that the rod mutant underwent abnormal early development since Sp7 when the carpel primordium initiated from the lemma side of floral meristem and stamens differentiated into anther and filament in the wild type.ROD gene was isolated by the map-based cloning approach and encodes a single C₂H₂ zinc finger protein localized in the nucleus.Phylogenetic analysis reveals that ROD protein is closely related to SUPERMAN in Arabidopsis.q RT-PCR assays indicated that ROD was expressed in the specific stages of flower development,without expression in the vegetative organs,such as root,culm and leaf.RNA in situ hybridization revealed that ROD was initially expressed in the third-whorl founder cells,then its expression was detected in the basal region of stamen primordia,namely the position of filament formation.Furthermore,ROD transcripts were also observed in the boundaries between adjacent stamen primordia.Since ROD is not expressed in anther and carpel,ROD affects their development in a non-cell-autonomous manner.Transcriptomic analysis showed that the rod mutation altered expression of several genes involved in cell proliferation,cell expansion,phytohormone signaling pathway and so on.By virtue of the DR5::VENUS marker,the auxin level was found to have no obvious change in rod mutant compared with wild type.Furthermore,we examined the genetic interactions between ROD and floral homeotic genes OsMADS3,OsMADS16,and OsMADS58 during flower development.The phenotype of double mutants showed that osmads3-4,spw1-1,and osmads58 were epistatic to rod except the enhanced defects in FM determinacy,respectively.In summary,this work not only reveals how the meristem maintenance gene genetically interacts with floral homeotic genes in specifying rice FM activity,it also provides new insights into the molecular mechanism of the floral organ size regulation.