Molecular Mechanism Of LATERAL FLORET 1 Inducing The "Three-florets Spikelet" In Rice(Oryza Sativa L.ssp.indica)

Rice?Oryza sativa L.?is an important food crop in the world and a model plant for studying functional genes of monocotyledons.Floral organ is the most complex organ structure in plants,and the development of it has served as one of the main model systems to study the genetic and molecular mechanisms that control organogenesis in plants.There are several reasons for this.Firstly,flowers contain the reproductive organs of angiosperms,which is the largest group of land plants,and thus are of pivotal importance for biological evolution.Secondly,flowers can eventually develop into food for humans and animals,and this ability contributes to its agricultural and economic.Hence,research on flowers has substantial translational potential.Lastly,although the structure of flowers and the organ types are largely conserved,flowers of different angiosperms do exhibit a great degree of variation in size,symmetry,organ number,organ arrangement as well as organ morphology.Therefore,the research on the molecular mechanism of floral organ development is of crucial importance.In the study,the LATERAL FLORET 1?LF1?gene was cloned,which invoved in lateral floret development of spikelet in rice.And the phynetype of lf1,the expression pattern and the fuction of LF1 were analysed.The results are as follows:1.lf1 developed lateral floretsIn the lf1 spikelet,no significant abnormalities were observed in the terminal floret and rudimentary glumes.However,the lateral florets contained inner floral organs grew obliquely in the axil of the upper and/or lower sterile lemma in an alternate phyllotaxy in lf1 spikelet.In these lateral florets,all types of inner floral organs were discovered,including pistil,lodicule,stamen and palea.During the early spikelet development stages,palea primordium was formed in the lateral florets and the initiation of stamen and pistil inner floral organ primordia started during the Sp7-Sp8 stage after the lemma and palea had closed in the terminal floret.Besides,the developmental stage of the lateral florets was delayed?approximately two Sp stages?compared with that of the terminal floret.2.Molecule mapping of LF1We employed cross between Xinong 1A and lf1 mutant.The phenotype of F₁population was similar to that of lf1 mutant,and the segregation ratio of mutant individuals and WT plants from F₂ progeny was a good fit to 3:1,which demonstrated that the lf1 trait was controlled by a single dominant gene.Next,the F₂ recessive single plant that hybridized with Xinong 1A and lf1 mutants was used as a mapping population to map the LF1 gene,and the LF1 gene was located at a 95.3-kb region between markers RM14281 and SNP-20 on the short arm of chromosome 3 finally.3.Cloning and Function analysis of LF1 geneA nucleotide substitution from C to T was identifed in annotated gene LOC_Os03g01890 in the lf1 mutant,causing a proline-192 to leucine-192 substitution.Further analysis showed that the mutation site was located in a putative target sequence for miRNA165/166.Next,we expressed the WT LOC_Os03g01890CDS-GFP and mutated LOC_Os03g01890CDS-GFP fusion genes under the control of the ubiquitin promoter in WT plants.In the transgenic plants that overexpressed mutated LOC_Os03g01890,the spikelets developed lateral florets,which was similar to the findings in lf1.In the transgenic plants overexpressed WT LOC_Os03g01890,the spikelets displayed normal morphology.Besides,in the transgenic plants of LOC_Os03g01890 RNAi,the spikelets displayed normal morphology.Therefore,these results suggested that LOC_Os03g01890 is LF1 gene.4.Expression Pattern of the LF1 gene and miRNA165/166By qRT-PCR analysis and GUS stain,LF1 expressed generally in young panicles and all floral organs during the heading stage.By in situ hybridization analysis,in WT spikelets,LF1 was expressed strongly in the FM centre domain and the adaxial domain of all the lateral organs,including the sterile lemma,lemma/palea,lodicule and stamen.In the lf1 spikelet,the expression domain of LF1 was expanded obviously.Firstly,the expression of LF1 was expanded from the FM centre domain to the cells located between the sterile lemma and palea,which might be precursors of the lateral FM.Next,LF1 was expanded from the adaxial domain to the abaxial domain of all the lateral organs.By in situ hybridization analysis,in WT spikelets,miRNA165/166 was expressed mainly in the margin domain of FM and abaxial domain of all the lateral organs,including the sterile lemma,lemma/palea,lodicule and stamen.In the lf1 spikelet,the expression pattern of miRNA165/166 was also similar to that of the WT,except that obvious expression of miRNA165/166 was detected in the lateral FM domain at the axil of the sterile lemma.5.Function analysis of LF1 proteinLF1 was found to encode a rice HD-ZIP III transcription factor.HD-ZIP III protein consists of four domains,including Homeodomain domain,Leu-zipper domain,START domain and MEKHLA domain.Phylogenetic analysis revealed that the angiosperm HD-ZIP III genes contained three clades,referred to as the REV,PHB/PHV and CNA/HB8 clades,and LF1/OsHB1 belonged to the REV clade.The subcellular localization analysis demonstrated that LF1 protein was localized in the nucleus.Furthermore,the transcriptional activation assay results suggested that LF1 is a transcriptional activator.6.LF1 Regulates?Floral?Meristem-related Genes to Initiate the Lateral FMBy qRT-PCR analysis,the expression level of OSH1 was increased significantly in the panicles of lf1 compared with the WT.Next,in situ hybridization analysis demonstrated that the expression pattern of OSH1 in the terminal floret of lf1 was generally similar to that of the WT,and a strong expression signal for OSH1 was detected in the lf1 lateral FM.Subsequently,chromatin immunoprecipitation?ChIP?assays were used to examine whether LF1 protein can bind directly to the OSH1 promoter.Chromatin isolated from young panicles of the mutated LF1-GFP?mLF1-GFP?transgenic plants and the WT were immunoprecipitated with an anti-GFP antibody and an anti-LF1 antibody respectively,and then subjected to qRT-PCR analysis.The results showed that both mLF1-GFP and WT LF1 protein cloud bound to four regions of the OSH1 promoter,which contained 1–6 conserved binding motifs?ATGAT?for the HD-ZIP III protein.These findings suggested that LF1 might regulate the OSH1 gene directly in both the lf1 and WT spikelet.Analysis by qRT–PCR also showed that the expression of the FM identity genes OsMADS6 and OsMADS15 was increased significantly in the panicles of lf1 compared with that in the WT.Next,the expression patterns of OsMADS6 and OsMADS15 were determined in detail by in situ hybridization.The results showed that clear expression signals of OsMADS6 and OsMADS15 were detected in the lf1 lateral FM from stages Sp5–Sp8.Thus,it is suspected that the FM identity genes OsMADS6 and OsMADS15might also involve in the initiation of lateral FM in lf1.7.LF1 Involved in the Evolution of“three-florets spikelet”in RiceLF1 may participate in the evolution of"three-florets spikelet"in two ways.The first possibility is that the lateral florets first degenerates into a sterile lemma under the regulation of G1,EG1,OsMADS34,NSG,ASP1,OsMADS1 and other genes,and then,the inner floral organs further degenerated under the action of the LF1 pathway.The second is the possibility that the inner floral organs of the lateral florets first degenerated under the action of the LF1 pathway,and then,the lemma of the lateral florets degenerated into sterile lemma under the control of G1,EG1,OsMADS34,NSG,ASP1,OsMADS1 and other gene,to form the wild-type spikelet.In addition,in future breeding efforts,we may be able to cultivate three-florets spikelet rice by combining the lf1 mutant with other mutants or transgenic plants?sterile lemma transformed into lemma?,to increase per spike.