Cloning And Functional Analysis Of FZP In Controlling Rice Inflorescence Branching

Common wild rice(Oryza rufipogon Griff.)is the wild ancestor of Asian cultivated rice(Oryza sativa L.).Compared with wild rice,marked morphological and physiological changes have found in the domesticated cultivars.Using an accession of common wild rice collected from Dongxiang County,Jiangxi Province(DXCWR)as a donor,and two high-yield cultivars 93-11(O.sativa ssp.indica)and C418(O.sativa ssp.japonica)as the recurrent parents,respectively,two sets of introgression lines(ILs)were developed.QTL analysis for 11 yield-related traits was conducted using these two ILs.The effects of these QTLs in two different genetic background was further evaluated.Based on the above results,a major pleiotropic locus(COSI)located on chromosome 7 was selected for further gene cloning and characterization.The major results were as follows:(1)QTLs for 11 agronomic traits including plant height(PH),number of tillers(NT),flag leaf width(FLW),number of primary branches(NPB),number of grains on primary branch(GPB),number of secondary branches(NSB),number of grains on secondary branch(GSB),grain length(GL),grain width(GW)and thousand-grain weight(TGW)in the two IL were mapped.A total of 118 QTLs were detected in the ILs derived from the corss between 93-11 and DXCWR and 99 were detected in the C418×DXCWR population,and 56 QTLs could be simultaneously detected in both genetic backgrounds and shared the same genetic effects.Many QTLs for yield-related traits were mapped to the same or adjacent genomic regions.Ferthemore,13 QTL clusters located in chromosome 1,3,4,6,7,8,9 and 11 were detected,implying that these regions may be the "hot spots" underlying genetic divergence between wild and cultivated rice functional genomes.(2)A major locus for NSB,GSB and GN located in chromosome 7(COS1)was consistently detected in the two genetic backgrounds.Further phenotypic observations of an introgression line(8IL50)harboring the COS1 locus from DXCWR showed that compared with that of the recurrent parent C418,NSB,GSB,GN,PH,FLW,GL and TGW in 8IL50 were all changed,suggesting that the COS1 locus showed pleiotropic effects.(3)COS1 was finely mapped to a 7.3-kb genomic region from a segregate population derived from the cross between C418 and 8IL50.Within this region,only the FRIZZY PANICLE(FZP)gene encoding an AP2/ERF domain containing protein was annotated according to the Nipponbare Genomic Database.Sequence analysis revealed that there was only a 4-bp deletion in the 7.3-kb region located at about 2.7-kb upstream of FZP start codon both in C418 and 93-11 when compared with DXCWR.The 4-bp deletion,which was located adjacent to a conserved auxin response factor binding element,may affect the binding affinities of OsARF6 to the FZP promoter,thereby decreasing the FZP gene expression.Further transgenic analysis suggested that higher expression level of FZP can induce less grain number by decreasing NSB and GSB.Both RT-PCR and RNA in-situ hybridization showed that FZP expression was mainly detected in young meristem with higher levels in 8IL50 than in C418,suggesting that FZP was an key regulator controlling rice branch and floret meristems development.Comparison of the sequence diversities of FZP among different wild and cultivated rice revealed that all of the cultivated rice harbored the 4-bp deletion and there was an about 150-kb selective sweep region surrounding the FZP gene in cultivated rice,suggesting that FZP was subjected to strong artificial selection.(4)The FZP associated proteins in vivo were identified by mass spectrometry analysis using the protein complex purified from the pUbi-Flag-FZP-GFP transgenic plant.Further GST pull-down,BiFC and Co-IP assays were performed to verify the interaction between FZP and a co-purified protein NAL1.A new NAL1 mutant allele(G257D,nal1-4)showing a significant decrease in NSB,GSB and GN was identified.Overexpressing wild type NAL1 CDS in nal1-4 fully complemented the phenotypic changes,suggesting that NAL1 is a positive regulator of NSB,GSB and GN.Both in vitro and in vivo degradation assays further revealed that NAL1,which encodes a trypsin-like serine and cysteine protease,could degrade FZP.Consistently,FZP protein level was over-accumulated in nal1-4 compared with wild type TQ.RNA-seq analysis also showed that NAL1 and FZP commonly regulated many downstream target genes,suggesting that NAL1 and FZP could function in the same genetic pathway in vivo.Further transgenic assays showed that down-regulating the expression of FZP in nal1-4 mutant could partially rescue the developmental defects found in nal1-4,including a significant increase in NSB,GSB and FLW,suggesting that FZP acts genetically downstream o fNAL1 to modulate rice development.(5)Finally,both up-regulated NAL1 expression and moderately down-regulated FZP expression in commercial variety ZH17 have potentials in enhancing rice grain yield.Taken together,these results suggested that NAL1 could enhance rice grain number and yield by degrading FZP.The cloning and functional characterizing of FZP not only addresses questions that are relevant to the molecular basis of rice evolution as well as grain production,but also provide new and valuable genetic resources for high-yield rice breeding program.