Map-Based Cloning And Functional Analysis Of The Rice Panicle Architecture Gene VPB1

The inflorescence architecture is a key factor affecting rice yield,which is mainly determined by the arrangement of the branches and flowers.The inflorescence meristem initiates primary branches in a spiral phyllotaxy,and spikelets are formed on these branches.This branching pattern is of great importance for constructing inflorescence architecture of rice.Here,we have studied a inflorescence mutant from rice T-DNA insertion mutant library,we designated this as verticillate primary branch 1(vpb1),this mutant exhibited the clustered primary branch phenotype and the length of rachis was reduced.We used map-based approach to locate a gene VPB1(LOC＿Os05g38120)that regulated panicle architecture,and through genetics,molecular biology and transcriptomics,the regulatory network of VPB1 gene regulating rice inflorescence development was analyzed.The main results are listed as follows:1.Phenotypic identification of vpb1 mutantIn contrast to the wild type inflorescence,the rachis length was shorter in vpb1 mutant,and the distance between the branch sites was shorter,which led to the primary branches clustered together,initiated in a verticillate manner,and the number of PBs increased,the number of SBs and the number of grains decreased.Morphological analysis indicated that the inflorescence meristem of vpb1 mutant was defective.The primary branch meristem was clustered on the inflorescence meristem,and the height of the whole inflorescence meristem was lower in vpb1 than in wild type.This data showed that the disordered phyllotactic pattern of vpb1 was caused by the disturbed initiation and arrangement of primary branch meristem.2.The cloning of VPB1 geneThe F2 population was constructed by crossing the vpb1 mutant with indica variety Dular,through phenotypic investigation and chi-square test,we found that VPB1 was a recessive single gene.We localized VPB1 on chromosome 5 through a map-based approach,the mutation phenotype and genotype were co-segregated.Genetic complementation and overexpression test showed that VPB1 gene could make vpb1 mutant phenotype return to wild type,moreover,the phenotype of the created crispr-vpb1 mutants confirmed that the loss of VPB1 gene function led to the verticillate primary branches phenotype of the vpb1 mutant.3.Molecular characteristics and expression patterns of VPB1 geneEvolutionary analysis and blast indicated that the VPB1 encoded a SKY BEL domain protein,which represents a BLH family member that is conserved in plants,and VPB1 was a homologous gene of the Arabidopsis PNY gene.We tested the subcellular localization and transcriptional activity of the VPB1 gene in rice protoplasts.These data illustrated that the VPB1 was a nuclear protein with transcriptional repressive activity.We characterized its expression pattern.qRT-PCR revealed that VPB1 was dominantly expressed in young panicle.RNA in situ hybridization further suggested that the VPB1 gene was observed in all reproductive meristems,especially where the branch meristems was formed.These evidences illustrated that the VPB1 contributed to establishing and maintaining meristem of rice.4.VPB1 interacts with OSH1 and OSH15 proteinsY2H,BiFC and pull-down assays verified the VPB1 interacts with OSH1 and OSH15 proteins in vivo and in vitro.The created crispr-osh1 mutant could not form a normal SAM,showed a phenotype of extremely dwarfing and lethal later,indicated that the OSH1 gene was very important for the formation and maintenance of SAM.The created crispr-osh15 mutant showed a phenotype of short plants,shortened internodes,and smaller panicle,indicated that the OSH15 gene was essential for internode elongation.Two double mutants,vpb1 osh1 and vpb1 osh15,were created,and their SAM development was severely defective,resulting in plant lethality.These evidences illustrated that VPB1,OSH1 and OSH15 can form heterodimers to regulate the development of meristems.5.The VPB1 gene involves complex regulatory pathways to control panicle developmentRNA-seq data analysis showed that VPB1 may be participated in multiple biological pathways.Especially in the process of inflorescence architecture and hormone signal transduction pathway played an important role.We performed q PCR to verify the DEGs participated in these two pathways,and the results were identical with the data from transcriptome sequencing.These results illustrated that VPB1 could influenced the differentiation ability of the inflorescence meristem(IM)through modulating the genes related to IM development and a variety of plant hormone pathways,and ultimately affect the development of panicle.6.VPB1 can directly regulate the expression of OsBOP1 geneThe online website plantpan3.0 was used to download the core sequences of BLH gene family combination,and the online tool MEME FIMO was used to search for these core sequences among the promoter sequences of all differential genes.We speculated the OsBOP1 acts downstream of VPB1.q PCR data demonstrated that VPB1 negatively regulated OsBOP1 expression,in situ hybridization analysis further verified this conclusion.The DLR and EMSAs confirmed that VPB1 could be combined with the promoter of OsBOP1 gene and supressed its expression.However,the created vpb1osbop1 double mutant did not revert to the wild-type phenotype,indicated that in addition to regulating the OsBOP1 gene,the VPB1 gene might also regulated other downstream target genes to control panicle development.In summary,we cloned a gene VPB1 that regulates panicle architecture and analyzed its function.VPB1 can interact with KNOX-like proteins,and by regulating genes involved in the development of inflorescence meristem and plant hormone pathways,and directly inhibiting the expression of OsBOPs genes,it can maintain the balance of the development of inflorescence meristem and lateral meristem,thereby ensuring the fine arrangement of panicle branches.Therefore,we determined that VPB1 is a key gene for the normal arrangement of panicle branches in rice.The results of this study have certain guiding significance for understanding the molecular mechanism of rice panicle branching pattern and improving rice panicle architecture.