Mechanism Analysis Of OsOFP22 Regulating Plant Morphology And Grain Shape By Gibberellin And Brassinosteroid In Rice(Oryza Sativa L.)

Ovate Family Proteins are plant specific transcription factors,playing important roles in regulating plant growth and development.The rice genome encodes 33 OFP proteins(OsOFP),OsOFP1,OsOFP2,OsOFP3,OsOFP8 and OsOFP19 have been reported to regulate plant morphology and grain shape in rice by participating in BR signaling.The Arabidopsis thaliana OFP(At OFP)was confirmed to function as a transcription suppressor,and the gibberellin(GA)synthase gene GA20ox1 is a target of At OFP1.In this study,we identified the function of OsOFP22 in inhibiting GA and BR signalings and regulating rice morphological development;we confirmed that OsOFP22 had transcriptional inhibition activity and interacted with GS9,which is an important transcription factor controlling grain shape;we found that CYP72A20 gene,encoding a Cytochrome P450 monooxygenase(CYP450),probably acts as a target of OsOFP22.Our results provide clues for further understanding the mechanism of OsOFP regulating rice growth and development,and lay a foundation for further study on the mechanism of OsOFP22 regulating the interaction between GA and BR.A new member of the CYP450 superfamily involved in regulating plant height and grain shape was identified,which provides a new target for rice molecular design and breeding.The main results are as follows:1.The haplotype and expression patterns of OsOFP22.Bioinformatics analysis showed that the of OsOFP22 protein contains DNA binding domain in N-terminal,and conserved OVATE domain in C-terminal.The promoter of OsOFP22 contains cis-acting elements related to hormone response and endosperm development.The haplotype analysis showed that OsOFP22 formed 7different haplotypes,and there were separations between the indica rice and japonica rice.GUS staining on the OsOFP22 pro::GUS transgenic rice showed OsOFP22 was mainly expressed in coleoptile,young root,panicles,sections,stem nodes and leaf sheath,but less expressed in the blade.In addition,the expression level of OsOFP22was significantly increased when the rice seedlings were treated with exogenous GA or BR,indicating that OsOFP22 is involved in GA and BR pathways.2.Phenotypic identification of OsOFP22-OE,OsOFP22-RNAi,and OsOFP22-Crispr/cas 9 transgenic rice.Morphological phenotypes of OsOFP22-OE,OsOFP22-RNAi and OsOFP22-Crispr/cas9(Osofp22)transgenic rice were investigated.OsOFP22-OE transgenic rice showed decreased plant height,rounder grain shape,and the decreased appearance quality of milled rice.However,no obvious morphological phenotypes were observed in both OsOFP22-RNAi and Osofp22 transgenic rice,suggesting that OsOFP22 may have functional redundancy with other OsOFP members.3.The GA-or BR-response of OsOFP22-OE transgenic rice.The GA-induced elongation of the 2^nd leaf sheath and the?-amylase activity induction were attenuated in OsOFP22-OE transgenic rice compared to WT.The contents of GA3 and GA4 increased and the expression level of GA biosynthesis genes were increased.The results of western blot showed that OsOFP22 overexpression inhibited the GA-induced degradation of SLR1,further inhibited the GA signaling in the transgenic rice.In addition,the BR-responsive phenotypes and the transcript level of BR-related genes of OsOFP22-OE transgenic rice were identified.The results showed that OsOFP22 overexpression inhibited BR signaling and decreased the expression levels of BR-related genes.4.Identification of the interaction between OsOFP22 and GS9 and its mechanism of regulating grain shape.The interaction of OsOFP22-GS9 was confirmed by yeast two-hybrid and bimolecular fluorescent complimentary(Bi FC)assays.OsOFP22 showed transcriptional inhibitory activity,and GS9 can regulate its transcriptional activity,that was confirmed by dual luciferase reporter assay.Haplotype 3 completely formed by japonica rice varieties has the smallest grain length,the largest grain width and the smallest length width ratio among all the haplotypes,while the 1000-grain weight did not change,indicating that the haplotype 3 is an important allele regulating round grain phenotype.5.Identification of CYP72A20,a target gene of OsOFP22.RNA-Seq results showed that the expression levels of several genes belonging to CYP72A subfamily of the CYP450 superfamily were changed in OsOFP22-OE plants.Subsequently,the yeast one hybrid assay and the LUC/REN assay carried out in rice protoplast showed that OsOFP22 could bind the promoter of CYP72A20 and inhibit the transcription of CYP72A20,which indicates that CYP72A20 probably functions as a target of OsOFP22.6.The functional analysis of CYP72A20.The CYP72A20-OE and CYP72A20-Crispr/cas9(cyp72a20)transgenic rice were constructed,and their morphological phenotypes were investigated.The results showed that the grain shape of CYP72A20-OE became longer,shorter and thinner than those of the WT,which was contrary to those of OsOFP22-OE.In addition,CYP72A20negatively regulated the plant height,with the semi-dwarf and higher plants of CYP72A20-OE and cyp72a20 transgenic rice respectively.The expression level of several GA-or BR-related genes were change in the transgenic rice.These results suggest that CYP72A20 regulates rice growth and development at least through GA and BR pathways.