Identification Of The Target Gene Of Ovate Family Protein Members And The Mechanism By Which It Regulates Brassinosteroid Responses In Rice

Brassinosteroids(BRs)are a class of plant specific steroid hormones.In rice,BR regulates multiple important agronomic traits.Ovate family proteins(OFP)are plant-specific transcription factors that regulate various aspects of plant growth and development.The OFP family proteins in rice(OsOFPs)play important roles in regulating plant architecture,grain shape,embryo sac development,and stress resistance.At least 5 members of OsOFPs have been reported to be involved in regulating BR signaling and regulating rice plant architecture and grain shape.It has been over 20 years since the first gene of the ovate family proteins(Solanum lycopersicum L.OVATE)was cloned.Here we report a novel rice gene(LOC-Os03g02470)as the target of OsOFP3,OsOFP19,and OsOFP22 through transcriptome sequencing(RNA-Seq),a series of biochemical experiments,as well as the construction of the transgenic rice and the analyses of its hormone response and morphological phenotypes.As its overexpression enhances the BR response in transgenic rice,it is named enhanced brassinosteroid response 1 in rice(OsEBR1).OsEBR1 interacts with the BR signaling components DWARF AND LOW-TILLERING(DLT)and Oryza sativa homeobox1(OSH1)and alleviates the transcription inhibitory activities of OsOFPs to its own coding gene by modulating the activity of DLT and OSH1,thereby maintaining basic BR activity in plants.This study discovered a new gene OsEBR1 as a target gene of OsOFP members to positively regulate BR response,and a new mechanism by which OsEBR1 regulates rice BR responses.The specific results are as follows:1.The bio-informatics analysis,protein subcellular localization,and the expression characteristics of OsEBR1The results from RNA-Seq of OsOFP22-OE young panicles showed that nearly2/3 of the differentially expressed genes did not have any functional annotation.Real time fluorescence quantitative PCR(RT-q PCR)results confirmed that the expression levels of OsEBR1 were upregulated more than 500 times in the OE lines.There are11 BR responsive elements in OsEBR1 promoter,and 11 SNP sites in its promoter and the coding sequence.One SNP in its coding region causes amino acid changes;Its alleles form 8 haplotypes,and haplotypes of indica and japonica segregates.Except for a homologue encoded by LOC＿Os06g15430 in rice,no homologue of OsEBR1 has been found in other species.There are 11 potential GSK3 phosphorylation sites in its amino acid sequence,and amino acid residues from 238 to 278 form an internally disordered region(IDR).The OsEBR1 protein is localized in the nucleus and cytoplasm,and forms large protein aggregates in the cytoplasm.OsEBR1 expression is tissue-specific and is highly expressed in the coleoptiles,stem nodes and ligules.The transcript level of OsEBR1 is response to BR induction.2.OsOFP members directly inhibit OsEBR1 transcription and the feedback upregulation of OsEBR1 expression in OsOFP22-OEThe results from yeast one-hybrid(Y1H),DNA-pull down,dual luciferase(Dual-LUC),and chromatin immunoprecipitation fluorescence quantitative PCR(Ch IP-q PCR)assays showed that OsOFP3,OsOFP19,and OsOFP22 bound to the OsEBR1 promoter and inhibited its transcriptional activity,and recognized the regions of-1200–-800 bp and-600–-200 bp.All three OsOFP members inhibited the activity of the OsEBR1 promoter to drive the expression of the reporter gene.OsBZR1,the core transcription factor of BR signaling,bound to the promoter of OsEBR1 through BR responsive elements and inhibits its transcription.In OsOFP22-OE,the OsBZR1 protein was attenuated and reduced the transcriptional inhibition of OsEBR1.The interaction between SLR1 and OsBZR1 also reduced the function of OsBZR1 to OsEBR1,ultimately leading to feedback upregulation of OsEBR1 expression in the OsOFP22-OE.3.OsEBR1 regulates BR responses and BR-related agronomic traits in riceWe constructed transgenic rice with OsEBR1 overexpression(OsEBR1-OE)and the knockout mutants by Crispr/cas9 technology.The homozygotes of OsEBR1loss-of-function mutants were lethal and the heterozygotes(Osebr1)were used for the functional analysis.The BR response assays,the expression pattern of BR-biosynthesis genes and the endogenous BR contents all confirmed that overexpression of OsEBR1 enhanced BR signal transduction and feedback inhibited BR biosynthesis.OsEBR1 is indispensable of BR responses in rice.OsEBR1 regulates plant height,flag leaf inclination,panicle morphology,and grain shape.OsEBR1 overexpression promotes cell division and inhibits cell expansion.Under physiological conditions,OsEBR1 contributes to BR promote GA accumulation and cell elongation.However,overexpression of OsEBR1 causes excessive activation of BR signaling,which in turn inhibits GA activity.The OsEBR1-OE grains are longer,wider,and has a higher 1000-grain weight than the WT,and the ratio of grain length to width of OsEBR1-OE is also increased.The enlarged spikelets of OsEBR1-OE were mainly determined by the increased cell volume.The Osebr1 is semi dwarfed,and the flag leaf inclination decreases and produces smaller spikelets.4.OsEBR1 interacts with and regulates the activities and protein accumulation patterns of DLT and OSH1Results from the yeast-two-hybrid(Y2H),fluorescent protein co-localization,split luciferase complementation(split-LUC),co-immunoprecipitation(co-IP),and bimolecular fluorescence complementarity(Bi Fc)assays demonstrated that OsEBR1 interacted with DLT and OSH1.OsEBR1 promoted the dimerization and transcriptional activity of DLT and inhibited those of OSH1.OsEBR1 maintains BR-induced DLT protein accumulation,while overexpression of OsEBR1 causes BR-induced OSH1 protein attenuation.In Osebr1,BR induces OSH1 protein accumulation.5.OsEBR1 alleviates the transcription inhibitory activity of OsOFP3 and OsOFP19 to its own coding geneThe results from Co-IP,DNA-pull down,and Dual-LUC assays demonstrated that OsEBR1 promoted the interaction between DLT-OsOFP3 and DLT-OsOFP19,while inhibited the interaction between OSH1-OsOFP3 and OSH1-OsOFP19.DLT alleviateed the inhibitory activity of OsOFP3 or OsOFP19 to the OsEBR1 promoter,while OSH1 promoted this inhibitory activity.OsEBR1 alleviates the transcription inhibitory activity of OsOFP3 and OsOFP19 to its own coding gene by enhancing the inhibitory function of DLT on OsOFP3 and OsOFP19 and inhibiting the promoting function of OSH1 on OsOFP3 and OsOFP19.