Molecular Analysis Of ZmIBH1-1 Regulating Plant Architecture In Maize

Maize,as one of the main three cereal crops,plays a key role on the food security worldwide.With the discordant between the needs from the fast increasing human population and the limited farming land becomes more and more prominent,to improve the yield of maize has been more and more pressing.Leaf angle,together with leaf length and leaf width,is a very important component of plant architecture,defining as the angle between the leaf midrib and the plant axis above it in maize.Leaf angle is an agronomic trait related to the planting density,ideal plant architecture holds an upright leaf angle,as upright leaf angle indicates a potential yield advantage through a higher planting density and a more efficient light capture through a better lower level canopy of plants,thus to an increased yield.Also leaf angle has been used as an important selection for high density planting in modern corn breeding,and planting density has increased from 30,000 per hectare at 1930s to 80,000 per hectare now,hybrids with upright leaves showed yield dominance in model simulation,field experiments and breeding practices.Traditional breeding method brought huge revolution of maize yield,while people know little about the mechanism underlying the leaf angle regulation.So understanding the regulation mechanism is very necessary,also it will not only help us to solve the fundamental question in plant science research,but also can help us to improve the breeding resources and contribute to our breeding practice.In this study,on the basis of preliminary primary mapping,utilizing inbred line Yu82 and inbred line Yu87-1 to construct fine mapping populations:BC3F1,BC4F1,BC5F1 and BC3F2,BC3F3,BC4F2,BC4F3,and BC5F2.The QTL qLA12-1 was located within the 14.07 kb interval between the newly developed markers SSR2-28/SSR2-35 on the second chromosome,and the candidate genes within the region was cloned through map-based cloning.At the same time,CRISPR-Cas9 mutagenesis method was used to preliminarily verify the function of IBH1 on leaf angle in the model system Setaria viridis.Through the DAP-Seq combined with RNA-Seq methods,we identified 23 genes may be involved in the molecular regulation of the size of maize leaf angle,revealing the molecular regulation networks involved in the regulation of maize leaf angle.The main conclusions are as follows:1.Using Yu82(erect leaves)and Yu87-1(extending leaves)to build fine mapping populations:BC3F2/BC3F3,BC4F2/BC4F3,and BC5F2/BC5F3.The main effect QTL qLA2-1 was fine mapped.The qLA2-1 interval is defined between the markers SSR2-28 and SSR2-35,and the target zone distance is reduced from 0.42 Mb to 14.37 kb,releasing a BAC(AC193307).There are two coding genes in the 14.37 kb region,namely GRMZM2G388823 and GRMZM2G535623.GRMZM2G388823 is orthologous to OsIBHl,a gene that regulates rice leaf angle,encoding a protein that consists of208 amino acids,and has typical bHLH conservation,so GRMZM2G388823 was named ZmIBHl-1 due to the presence of its other homologous gene in the entire maize genome.2.Quantitative real-time PCR showed that the expression level of ZmIBHl-1 in leaves was higher in Yu82 and Yu87-1-NILthan that ofYu87-1,also the CRISPR/Cas9 mutagenesis system knocked out the homologous gene SevirIBHl in the system Setaria viridis.After the knockout of SevirIBHl,the phenotype of the flag leaf angle changed significantly,which proved that SevirIBHl regulated leaf angle negatively.3.Using MEME programe,results showed ZmIBH1-1 binding motifs E-box NNCAAGTNG CANGTN,also found three new binding sites:CTTCGNN,GGNGGAGA and CCTNNG.Further analysis revealed that 942 target genes overlapped in two biological replicates,of which 344 target genes locatd on promoters.These genes mainly involved hormone response,organ development,cell composition,and anatomical development.Developmental processes and other biological processes.The bioinformatics analysis and gene function annotation results indicate that 81 genes involved in cell division,differentiation,cell wall metabolism,and auxin,brassinosteroids,cytokinins,gibberellin synthesis,and signal transmission may affect maize leaf angle formation.4.DAP-Seq combined with RNA-Seq analysis showed 23 genes may be involved in the mazie leaf angle regulation,for example,genes involved in cell wall metabolism(GRMZM2G180160,GRMZM2G004435,GRMZM5G813143,GRMZM2G048821,GRMZM2G074850,GRMZM2G427337 and GRMZM2G134256),Genes involved in cell proliferation(GRMZM2G317652 and GRMZM2G339563),genes involved in auxin synthesis and response(GRMZM2G022212,GRMZM2G168502,AC210199.4_FG003,and GRMZM2G342281),genes involved in BR response(GRMZM2G104342,GRMZM2G012861,GRMZM2G136106,GRMZM2G057329,and GRMZM2G327595),genes involved Genes that respond to cytokinin(GRMZM2G009344,GRMZM2G335618,and GRMZM2G365374),genes involved in GA signaling(GRMZM2G365374),and genes involved in the gibberellin metabolism(GRMZM2G033413),these genes provide us a primary maize leaf angle molecular networks which gene ZmIBHl-1 involved in.5.Histological analysis revealed that compared with Yu87-1,NIL has a well-developed support structure at the distal end,described as enlarged vascular bundles,a high level of lignification,more layer of scelerenchyma cells.ZmIBHl-1 directly regulates genes related to cell wall metabolism,cell division and differentiation,and phytohormone metabolics,to affect the proliferation,differentiation,and size of vascular bundles at the distal epidermal end of the ligular region to create the leaf angle difference,and finally resulted the difference of maize palnt architecture.