| Brassinosteroids(BRs)are an important group of growth-promoting hormones found throughout the plant kingdom.Genetic studies demonstrated that BRs play essential roles during nearly all phases of plant growth and development.BRs play vital roles in several valuable agronomic traits such as plant height,architecture,and kernel size of maize and other crops.However,few genes involved in BR biosynthesis or signaling have been identified in maize so far.Copine are a group of highly conserved gene family ubiquitously found in various eukaryotes including human,mammals and plants.The conserved nature of copine in different organisms suggest that they play important roles in common biological pathways,which is supported by emerging studies.In plants,the study of copine gene was first carried out in the model plant Arabidopsis.Because the mutant of copine gene showed a dwarf morphology,they were also named BONZAI(Bonzai).BONZAI have been reported to be negative regulators for several decades,because the bon1-1 mutant exhibited autoimmune phenotypes.However,the exact and conserved function of BONZAI genes remains unclear.Here,by studying the BONZAI homologous genes in maize,we discovered that the loss of ZmBON1 function led to a dwarf morphology not caused by autoimmunity but by deficient BR signaling.Consistently,the Arabidopsis bon1-1bon2-2bon3-3pad4-1 mutant showed compromised sensitivity to BR treatment,indicating that the biological function of BONZAI in BR signaling are conserved among species.Further study indicates that BON proteins are required for BRI1–SERKs protein complex formation and the subsequent phosphorylation cascades events.The main conclusions and results are as follows.1.Loss of ZmBON1 function leads to a dwarf phenotype in maize.In this study,we screened the maize genome,and only two genes ZmBON1 and ZmBON3,which show close homology to BON1 and BON3 in Arabidopsis,were identified.We utilized clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated nuclease 9(Cas9)-mediated gene editing to generate Zmbon1 and Zmbon3 single mutants and Zmbon1bon3 double mutants.All Zmbon1 mutant lines showed drastic dwarf morphology,while none of the Zmbon3 single mutants showed obvious differences from wild-type KN5585.The Zmbon1bon3 double mutants exhibited enhanced dwarfism compared to the Zmbon1 mutants.Meanwhile,we also identified two EMS mutants in the B73 background for ZmBON1 and ZmBON3,namely Zmbon1-7 and Zmbon3-7,respectively,wherein point mutations were predicted to result in truncated proteins.Like the Zmbon1 and Zmbon3 mutants in the KN5585 background,Zmbon1-7 showed obvious dwarfism,while Zmbon3-7 showed no morphological differences from the wild-type B73.2.The dwarfism of Zmbon1 is not caused by autoimmunity in maize.We observed no necrotic lesions on the leaves of Zmbon1-1,and analyzed reactive oxygen species(ROS)accumulation,which revealed no significant differences among KN5585 and Zmbon1-1.We observed no significant differences in ZmPR1 or ZmPR5 gene expression in Zmbon1-1 compared to KN5585.Moreover,the level of the defense phytohormone salicylic acid(SA)did not change in the Zmbon1-1.We also carried out transcriptome deep sequencing(RNA-seq)of the wild-type KN5585 and Zmbon1-1,while defense-related terms were not significantly enriched.3.ZmBON1 is involved in BR signaling in maize.Morphological analysis showed that Zmbon1-1 exhibited a variety of characteristics of BR-defective mutants including shorter internodes,abnormal leaf angle,and smaller kernels.We performed a root growth inhibition assay by BL treatment and found that the Zmbon1-1 in the KN5585 background and Zmbon1-7 in the B73 background both showed reduced BL sensitivity.Moreover,the maize BR biosynthetic marker genes,ZmBRD1 and ZmCPD,were significantly upregulated in Zmbon1-1.4.BON’s involvement in BR signaling is conserved in the dicot Arabidopsis.The root inhibition assay showed that bon1-1bon2-2bon3-3pad4-1 also exhibited a dramatically insensitive to BL.In addition,the bon1-1bon2-2bon3-3pad4-1 mutant displayed significantly shorter hypocotyls than the pad4-1.Analysis of the phosphorylation status of BZR1 by western blot,displayed that the levels of dephosphorylated BZR1 increased less in bon1-1bon2-2bon3-3pad4-1compared to that of pad4-1.Based on the RNA-seq data,we found that genes in the‘brassinosteroid biosynthesis’ pathway are most significantly enriched among DEGs between pad4-1 and bon1-1bon2-2bon3-3pad4-1 upon e BL treatment.Furthermore,the transcriptional level of several BR-responsive genes showed significantly different between pad4-1 and bon1-1bon2-2bon3-3pad4-1 by RT-q PCR.5.BON1 interacts with BR receptor BRI1 and co-receptor SERKs.Yeast two-hybrid(Y2H)assay indicated that BON1 physically interacts with the co-receptor SERK1,SERK2,and SERK4.A split luciferase complementation assay(LCA)and co-immunoprecipitation(Co-IP)assay showed that BON1 interacts BRI1 and SERK1/2/4 in vivo.In addition,Y2 H and LCA assays indicated that ZmBON1 interacts with ZmSERK4 in vitro and vivo.6.BONZAI are critical for the formation of the BRI1-SERKs complex and reciprocal phosphorylation.BRI1 co-immunoprecipitated with SERK3/4 after BL treatment was dramatically less abundant in bon1-1bon2-2bon3-3pad4-1 than in pad4-1.By quantitative phosphoproteomics sequencing and analysis,we found that the phosphopeptides from several known BR signaling components including BRI1,SERKs,BKI1,BSK5/8,BSL2,and BSL3 were dramatically reduced in bon1-1bon2-2bon3-3pad4-1 upon BL treatment.To confirm the data from the quantitative phosphoproteomics analysis,we detected the abundance of phosphorylated BRI1 and SERKs in pad4-1 and bon1-1bon2-2bon3-3pad4-1 seedlings from mock and BL treatment,which was consistent with the results of the quantitative phosphoproteomics.This study advances the knowledge on BR signaling and provides an important target for optimizing valuable agronomic traits,it also opens a way to study steroid hormone signaling and copine proteins of eukaryotes in a broader perspective. |
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