Single-Cell Transcriptome and Network Analyses Unveil Key Transcription Factors Regulating Mesophyll Cell Development in Maize

Abstract

Background: Maize mesophyll (M) cells play important roles in various biological processes such as photosynthesis II and secondary metabolism. Functional differentiation occurs during M-cell development, but the underlying mechanisms for regulating M-cell development are largely unknown. Results: We conducted single-cell RNA sequencing (scRNA-seq) to profile transcripts in maize leaves. We then identified coregulated modules by analyzing the resulting pseudo-time-series data through gene regulatory network analyses. WRKY, ERF, NAC, MYB and Heat stress transcription factor (HSF) families were highly expressed in the early stage, whereas CONSTANS (CO)-like (COL) and ERF families were highly expressed in the late stage of M-cell development. Construction of regulatory networks revealed that these transcript factor (TF) families, especially HSF and COL, were the major players in the early and later stages of M-cell development, respectively. Integration of scRNA expression matrix with TF ChIP-seq and Hi-C further revealed regulatory interactions between these TFs and their targets. HSF1 and COL8 were primarily expressed in the leaf bases and tips, respectively, and their targets were validated with protoplast-based ChIP-qPCR, with the binding sites of HSF1 being experimentally confirmed. Conclusions: Our study provides evidence that several TF families, with the involvement of epigenetic regulation, play vital roles in the regulation of M-cell development in maize.

Network and epigenetic characterization of subsets of genes specifically expressed in maize bundle sheath cells

“…Characterization of transcriptomic regulatory network can provide comprehensive insights into how hub TFs coordinate with key genes to result in functional divergences between two cell types. The single M-cell RNA-seq related network analyses revealed distinct roles of COL8 and HSF1 in the regulation of genes involved in the early and late leaf development [16] , [24] . However, the single BS-cell RNA-seq related network analyses are still unavailable since it is difficult to collect enough single BS cells for the single cell RNA sequencing [16] , [25] , partially due to technique limitation and the structural nature of BS cells.…”

“…The single M-cell RNA-seq related network analyses revealed distinct roles of COL8 and HSF1 in the regulation of genes involved in the early and late leaf development [16] , [24] . However, the single BS-cell RNA-seq related network analyses are still unavailable since it is difficult to collect enough single BS cells for the single cell RNA sequencing [16] , [25] , partially due to technique limitation and the structural nature of BS cells. Here, we for the first time conducted network analyses by analyzing bulk BS and M cell RNA-seq data.…”

“…It has been documented that Dof TFs play both active and repressive roles in the regulation of several C 4 gene expression like PPDK and PEPC [14] , [15] . It has been reported that some of TF genes are preferentially expressed and function in BS cells [8] , [9] , [16] . Some of bHLH family can preferentially bind to NADP-ME and PEPC .…”

“…Similarly, it has been reported that TFs in the MYB-MYC module can bind to the promoter CREs of some genes, resulting in their preferential expression in bundle sheath of Arabidopsis [23] . Furthermore, functions of TFs in the regulation of cell-dependent gene expression have been comprehensively characterized through regulatory network analyses using single M cell RNA-seq data, leading to discoveries that several CO-like TFs possibly play dominant roles in leaf development and photosynthesis for M cells in maize [16] , [24] . However, BS cell-related regulatory networks are still not completely characterized, especially it is hard to be achieved at a single cell level due to technique limiting for isolation of enough BS cells for sequencing as compared to single M cells [16] , [25] .…”

“…Furthermore, functions of TFs in the regulation of cell-dependent gene expression have been comprehensively characterized through regulatory network analyses using single M cell RNA-seq data, leading to discoveries that several CO-like TFs possibly play dominant roles in leaf development and photosynthesis for M cells in maize [16] , [24] . However, BS cell-related regulatory networks are still not completely characterized, especially it is hard to be achieved at a single cell level due to technique limiting for isolation of enough BS cells for sequencing as compared to single M cells [16] , [25] . To address this question, we here conducted WGCNA-related co-expression assays using bulk M and BS cell RNA-seq data sets and identified a co-expression module containing genes highly expressed in BS cells instead of M cells.…”