Deciphering The Transcriptional Regulatory Mechanisms Of C₄ Photosynthetic Genes Using Multidimensional Omics Data

Higher plants can be divided into C₃ plants,C₄ plants and CAM（Crassulacean Acid Metabolism）plants according to their photosynthetic carbon assimilation types.C₄ plants evolved from C₃ plants and have higher photosynthetic,water and nitrogen use efficiency than C₃ plants.The typical C₄ Kranz anatomy consists of two types of photosynthetic cells:bundle sheath（BS）cells form a ring around the vascular tissue,and mesophyll（M）cells form a ring around them.The specific localization and expression of C₄ photosynthetic related genes in two kinds of cells is the key to the CO₂ concentration mechanism and reduction of photorespiration for ensuring the efficiency of C₄ photosynthesis.To better understand the transcriptional regulation mechanism on C₄ photosynthetic related genes,we performed transcriptome（RNA-Seq）,chromatin accessibility（ATAC-Seq）,histone modification（Ch IP-Seq）and whole genome DNA methylation（WGBS-Seq）sequencing using BS and M cells isolated from leaves of C₄ species Zea mays.And we compared the data of Z.mays,Sorghum bicolor and Setaria italica for identifying the conserved differentially expressed genes and regulatory elements across C₄ species.The genes that may participate in C₄ photosynthesis were predicted using multiomics data and comparative genomics.This project provides a theoretical basis for further analysis of the regulation mechanisms for C₄ photosynthetic gene expression.The main results are as follows:1.Differential gene expression between BS and M cells in maize is regulated by the chromatin state of gene flanking regionsThe overall gene expression level is correlated with the intensity of chromatin modification around the gene.The expression level of differentially expressed genes between BS and M cells is also affected by chromatin status.And the combination of multiple chromatin features leads to a tight regulation of the cell type-preferentially gene expression.2.The ACRs affect cell type-preferentially expression of genesThis project found that not only the ACRs proximal to the genes（p ACRs）but also the distal ACRs（d ACRs）are determinants of cell type-preferentially gene expression.And there are usually differences in histone modification and DNA methylation modification around the differential ACRs between BS and M cells.3.Dof and WRKY transcription factor families affect the gene differential expression pattern between maize BS and M cellsWe identified the transcription factor binding motifs significantly enriched in the accessible chromatin region of BS and M cells,e.g.AAAG for BS cells and TGACY for M cells.Their corresponding transcription factors Dof and WRKY family members also have the same cell type-preferentially gene expression pattern.In addition to the Dof and WRKY families,G2-like,TCP and C2H2 transcription factor families are differentially expressed,while TCP and ERF are differentially bind between BS and M cells.4.Conservation of gene expression and accessible chromatin regions between BS and M cells across C₄ speciesUsing RNA-Seq and ATAC-Seq data,563 BS and M differentially expressed genes and303 differentially chromatin accessibility associated genes were identified across Z.mays,S.bicolor and S.italica,and 60 of these genes overlapped.Conserved genes among C₄ species are significantly enriched in the process of transcriptional regulation,suggesting that several master regulators may be selected in parallel in multiple species during the evolution of C₄photosynthesis.5.Construction of conserved gene regulatory network of C₄ speciesThirty-six transcription factors with conservative expression pattern and known binding motifs were used to identify binding sites in the differentially ACRs between BS and M cells to construct the gene expression regulatory network.We found 20 hub genes in the gene expression regulatory network and predicted the upstream core transcription factors of C₄related gene.6.Mining of C₄ related genes and regulatory sequencesBased on the epigenetic modification status and the conservation of expression patterns among C₄ species,we predicted a group of genes with unknown function as potential key C₄genes for CRISPR/Cas9 gene editing.And we the preliminarily verified that the conserved BS-specific ACRs in C₄ species can initiate the cell type-specific expression of GUS reporter gene.In summary,our findings provide deep insight into the architectures associated with C₄gene expression and provide new clues for further verifying the gene expression regulation mechanism present in C₄ species.