Knowledge of the transcription factor binding landscape (TFBL) is necessary to analyze gene regulatory networks for important agronomic traits. However, a low‐cost and high‐throughput in vivo chromatin profiling method is still lacking in plants. Here, we developed a transient and simplified cleavage under targets and tagmentation (tsCUT&Tag) that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction. Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP‐seq. Furthermore, we developed a strategy combining tsCUT&Tag with machine learning, which has great potential for profiling the TFBL across plant development.

Background Maize (Zea mays L.) is one of the most important crops worldwide. Although sophisticated maize gene regulatory networks (GRNs) have been constructed for functional genomics and phenotypic dissection, a multi-omics GRN connecting the translatome and transcriptome is lacking, hampering our understanding and exploration of the maize regulatome. Results We collect spatio-temporal translatome and transcriptome data and systematically explore the landscape of gene transcription and translation across 33 tissues or developmental stages of maize. Using this comprehensive transcriptome and translatome atlas, we construct a multi-omics GRN integrating mRNAs and translated mRNAs, demonstrating that translatome-related GRNs outperform GRNs solely using transcriptomic data and inter-omics GRNs outperform intra-omics GRNs in most cases. With the aid of the multi-omics GRN, we reconcile some known regulatory networks. We identify a novel transcription factor, ZmGRF6, which is associated with growth. Furthermore, we characterize a function related to drought response for the classic transcription factor ZmMYB31. Conclusions Our findings provide insights into spatio-temporal changes across maize development at both the transcriptome and translatome levels. Multi-omics GRNs represent a useful resource for dissection of the regulatory mechanisms underlying phenotypic variation.

Tassel branch number is a key trait that contributes greatly to grain yield in maize (Zea mays). We obtained a classical mutant from maize genetics cooperation stock center, Teopod2 (Tp2), which exhibits severely decreased tassel branch. We conducted a comprehensive study, including phenotypic investigation, genetic mapping, transcriptome analysis, overexpression and CRISPR knock-out, and tsCUT&Tag of Tp2 gene for the molecular dissection of Tp2 mutant. Phenotypic investigation showed that it is a pleiotropic dominant mutant, which is mapped to an interval of approximately 139-kb on Chromosome 10 harboring two genes Zm00001d025786 and zma-miR156h. Transcriptome analysis showed that the relative expression level of zma-miR156h was significantly increased in mutants. Meanwhile, overexpression of zma-miR156h and knockout materials of ZmSBP13 exhibited significantly decreased tassel branch number, a similar phenotype with Tp2 mutant, suggesting that zma-miR156h is the causal gene of Tp2 and targets ZmSBP13 gene. Besides, the potential downstream genes of ZmSBP13 were uncovered and showed that it may target multiple proteins to regulate inflorescence structure. Overall, we characterized and cloned Tp2 mutant, and proposed a zma-miR156h-ZmSBP13 model functioning in regulating tassel branch development in maize, which is an essential measure to satisfy the increasing demands of cereals.

HOX32, a member of the HD-ZIP III family, functions in the leaf morphogenesis and plant photosynthesis. However, the regulatory mechanism of HOX32 in maize has not been studied and the regulatory relationship in photosynthesis is unclear. We conducted a comprehensive study, including phylogenetic analysis, expression profiling at both transcriptome and translatome levels, subcellular localization, tsCUT&Tag, co-expression analysis, and association analysis with agronomic traits on HOX32 for the dissection of the functional roles of HOX32. ZmHOX32 shows conservation in plants. As expected, maize HOX32 protein is specifically expressed in the nucleus. ZmHOX32 showed constitutively expression at both transcriptome and translatome levels. We uncovered the downstream target genes of ZmHOX32 by tsCUT&Tag and constructed a cascaded regulatory network combining the co-expression networks. Both direct and indirect targets of ZmHOX32 showed significant gene ontology enrichment in terms of photosynthesis in maize. The association study suggested that ZmHOX32 plays an important role in regulation of plant architecture. Our results illustrate a complex regulatory network of HOX32 involving in photosynthesis and plant architecture, which deepens our understanding of the phenotypic variation in plants.