Mapping-based Cloning And Functional Analysis Of LF2 And LF3 Genes In Rice (Oryza Sativa L.ssp.indica)

Spikelet is a unique inflorescence unit of grasses such as rice（Oryza sativa L.）,and grain number per panicle is one of the key factors for the yield of grasses.The number and length of branches,spikelet density on branches,and the number of florets in spikelet are all important factors affecting the grain number per panicle.As the ideal model plant of monocot,it is of great significance to illustrate the genetic model of flower development by study the flower/spikelet development of rice.In recent years,preliminary regulatory networks of rice flower/spikelet development have been made.However,it is still necessary to explore more flower/spikelet mutants and illustrate the regulatory mechanisms of related genes to improve the regulatory networks,so as to provide theoretical basis and new breeding method for molecular breeding of rice and other plants.In this study,we identified two lateral floret development mutants lateral floret2（lf2）and lf3 from the EMS（ethyl methane sulfonate）mutagenesis library of XIDA 1B（Indica）.And the phenotype analysis of the two mutants,mapping cloning and function analysis of LF2 and LF3 were studied.The main results were as follows:1.Molecular mechanism of LF2 regulationg lateral floret development1.1 lf2 mutant developed lateral floretsThe wild type spikelets only developed one terminal floret which contains four whorls of floral organs.In the lf2 mutant,the spikelets had elongated rudimentary glumes and lemma-like sterile lemma.Some spiklets generated lateral floret/spikelets at the axils of the steile lemma and/or rudimentary glumes.The lateral florets contained normal floral organs such as lemma,palea,lodicule,stamen and pistil.During the early development stage,lateral meristems were differentiated at the axis of sterile lemma and/or rudimentary glumes.And the expresson of the meristem identity genes OSH1 and Os MADS15 was significantly upregulated in the lf2 panicles.In situ hybridization results showed that the expression pattern of OSH1 and Os MADS15 in the terminal floret of lf2 was consistent with that of the wild type,but there were obvious ecotopic expression signals at the axix of sterile lemma and rudimentary glumes where lateral meristem formed.These results indicated that the ectopic expression of meristem related genes OSH1 and Os MADS15 induced the formation of lateral meristems.1.2 Identification and transgenic confirmation of LF2By BSA method,the LF2 gene was localized on the long arm of chromosome 7.Sequence analysis showed that the first base of the fourth intron of LOC＿Os07g44210occurred a T-A insititution,which caused disordered recognition of intron during m RNA splicing and then formed multiple meaningless transcripts.Complementary test and CRISPR/Cas9 test results proved that the LOC＿Os07g44210 was the LF2gene.1.3 Expression pattern analysis of LF2 geneqRT-PCR results showed that LF2 gene was strongly expressed in all tissues（root,stem,leaf,sheath and inflorescence）of rice.And LF2 gene was strongly expressed in the sterile lemma,but weakly expressed in other floral organs.In situ hybridization results showed that LF2 gene was strongly expressed in spikelet/flower meristems,and also expressed in four whorls of floral organ primordia of rice inflorescence.1.4 LF2 gene encodes a SNF2 family chromatin remodeling factor that localized in the nucleusThe LF2 gene encodes a SMACAL1 subfamily chromatin remodeling factor of the SNF2 family.LF2 protein contains a DEXDc domain and a HELICc domain that could bind ATP and DNA.The results of subcellular localization and GFP fluorescence observation of LF2-GFP fusion protein in transgenic plants showed that LF2 protein was localized in the nucleus,and the dual luciferase reporter assay showed that LF2had no significant transcriptional activation activity.1.5 LF2 interacts with nuclear factors to form a complex to regulate the expression of downstream genesThe pGBKT7-LF2 was used as bait to screen the rice transcription factor library（Shanghai OE Biotech Co.,Ltd.）.Six possible interacting proteins such as nuclear factor Os NF-YA3 were obtained.The Y2H assay and Bi FC experiments showed that LF2 could interact with Os NF-YA3.The spikelet of Os NF-YA3^cas9（ZH11,Japonica）transgenic plant exhibited similar phenotype to that of lf2 mutant.Further interaction screening among LF2,Os NF-YB and Os NF-YC proteins were carried out.The result showed that there were interactions between LF2 and Os NF-YB5 and Os NF-YC1/4/6/7,suggesting that Os NF-YA3 may form heterotrimer with Os NF-YB5and Os NF-YC/4/6/7,and interact with LF2 to regulate the expression of downstream genes.1.6 LF2 gene controls sterile lemma development by regulating G1 expressionChIP-Seq was carried out in the wild-type and lf2 mutant panicles using a H3K27me3 antibody and then association analysed with RNA-Seq data.The results showed that the expression of G1 in the lf2 mutant was significantly down regulated with the increase of H3K27me3 level.In the lf2+g1 double mutant,the rudimentary glume was elongated to some degree,the sterile lemma obtained lemma identity and was longer than that of lf2 and g1 single mutant,indicating that LF2 may positively regulate the expression of G1 by influencing the H3K27me3 levels to regulate the development of sterile lemma.1.7 LF2 gene is involved in auxin signaling pathway and affects spikelet developmentKEGG analysis of RNA-Seq showed that most of the differential genes in lf2mutants involved in the plant hormone signal transduction pathway,especially the auxin pathway.The endogenous auxin content was significantly reduced in lf2 mutants,and the expression of auxin synthesis genes Os YUCCA2 and Os YUCCA4,auxin transport gene Os PINs and auxin response gene Os ARFs were down regulated in lf2.The results suggesting that LF2 gene may be involved in the auxin synthesis pathway,affecting the auxin content distribution in spikelets,and ultimately affecting the expression of meristem related genes OSH1 and Os MADS15,thus maintaining the normal development of spikelets.2.Molecular mechanism of LF3 regulating lateral floret development2.1 lf3 mutant formed lemma-like sterile lemma and lateral floretsThe wild-type spikelet sterile lemma is significantly smaller than the lemma and has a smooth surface.lf3 mutants generated elongated sterile lemma with characteristic of lemma.Axis of some sterile lemma developed lateral florets with complete four-whorl floral organ,and most of the lateral florets could finally form a grain.In a few cases,both sides of the sterile lemma developed lateral florets to form a"three-floret spikelet",which finaly generated three grains.2.2 Mapping based cloning and transgenic verification of LF3 geneIn previous studies,LF3 gene was localized on the long arm of chromosome 7between markers RM3555 and CY7-3.In this study,sequencing analysis of genes within the mapping regeion showed that a C-A substitution was occurred on the 98th base of the the AP2/ERF family transcription factor LOC＿Os07g47330/FZP.This base substitute led the 33^th codon changed into termination codon,resulting in premature termination of translation.Inflorescence of LOC＿Os07g47330^cas9 knockout plants continuly generated sub-branches,and could not form spikelet,which was similar to the inflorescence of lf3 mutant,proving that LOC＿Os07g47330 is just the LF3 gene.2.3 Expression pattern of LF3 geneqRT-PCR results showed that LF3 gene was almost not expressed in vegetative tissues,but was strongly expressed in inflorescence,and the expression level was significantly decreased with inflorescence development and elongation.In floral organs,LF3 gene was expressed the highest in the sterile lemma and was also expressed in the lodicule and pistil.The results of in situ hybridization showed that LF3 was highly expressed in the meristem and sterile lemma primordia.2.4 LF3 gene encodes an AP2/ERF proteinLF3 encodes an AP2/ERF family transcription factor and is homologous to the BD1 in maize.LF3 and its homologous proteins in gramineae are highly conserved and contain a typical AP2/ERF domain.At the C-terminal of LF3 protein located an acidic domain,which makes LF3 protein has a strong transcriptional activation activity.2.5 LF3 interacting proteins screeningThe LF3 truncated protein PGBKT7-LF3^1-244 was used as bait to screen the cDNA library of rice panicle to find the interaction protein of LF3.And 20 proteins including Os MADS1,NAL9,nuclear factor NF-YC6 and NF-YC7 and 3 chromatin remodeling complex associated proteins（Os02g0723700,Os11g0545600,Os02g0122000）were selected,which may interact with LF3 and participate in the growth and development of rice.2.6 LF3 gene may regulate rice flower/spikelet development through plant hormone signaling pathwayThe RNA-Seq analysis of lf3 mutants showed that the differential genes had the highest enrichment in the plant hormone signaling pathway,which contained most genes in the auxin synthesis pathway and the auxin signal response pathway.In addition,the auxin content of lf3 mutant was decreased compared with that of the wild type,and the expression levels of Os YUCCA1 and Os YUCCA6 genes were significantly decreased in lf3,suggesting that LF3 gene may affect the development of rice flower/panicle by regulating auxin synthesis.