Genome-wide characterization of cis-acting DNA targets reveals the transcriptional regulatory framework of opaque2 in maize.

Chaobin Li,Weiwei Qi,Bing Mei,Zhenyi Qiao,Yue Yuan,Yuanping Tang,Han Xiao,Yuanda Lv,Han Zhao,Xi Yang,Qian Wang,Rentao Song

doi:10.1105/tpc.114.134858

Abstract

Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we studied the transcriptome of o2 mutants using RNA sequencing (RNA-Seq) and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). The RNA-Seq analysis revealed 1605 differentially expressed genes (DEGs) and 383 differentially expressed long, noncoding RNAs. The DEGs cover a wide range of functions related to nutrient reservoir activity, nitrogen metabolism, stress resistance, etc. ChIP-Seq analysis detected 1686 O2 DNA binding sites distributed over 1143 genes. Overlay of the RNA-Seq and ChIP-Seq results revealed 35 O2-modulated target genes. We identified four O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16- and 18-kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. We built a hierarchical regulatory model for O2 that provides an understanding of its pleiotropic biological effects.

Highlights

Opaque2 (o2) is a nonlethal maize (Zea mays) seed mutant that has been used as a classic genetic marker for chromosome 7 since the early part of the last century (Emerson et al, 1935). o2 kernels contain over 70% higher lysine contents than wild-type maize kernels (Mertz et al, 1964), thereby significantly improving the nutritional value of the grain
27-kD g-zein genes are not downregulated by this mutation, an O2-like box containing the ACGT core sequence is found in the promoter region (Wu and Messing, 2012), but it remains unclear whether O2 directly regulates these zein genes
To establish the regulatory function of the O2 gene, RNA sequencing (RNA-Seq) analysis was performed with total RNA extracts obtained from wild-type and o2 endosperms 15 d after pollination (DAP; see Methods; Supplemental Figure 1)

Summary

Introduction

Opaque (o2) is a nonlethal maize (Zea mays) seed mutant that has been used as a classic genetic marker for chromosome 7 since the early part of the last century (Emerson et al, 1935). o2 kernels contain over 70% higher lysine contents than wild-type maize kernels (Mertz et al, 1964), thereby significantly improving the nutritional value of the grain. O2 activates transcription by binding conserved DNA motifs in its target genes, most of which contain an ACGT core sequence named the O2-box (Schmidt et al, 1992; Muth et al, 1996). We used RNA sequencing (RNA-Seq) to identify protein-coding and noncoding RNAs that are differentially expressed in wild-type and o2 endosperms to build a comprehensive and genome-wide regulatory framework for O2. These data indicate O2 significantly affects expression of 1605 genes and 384 long, noncoding RNAs (lncRNAs). The additional target genes were confirmed and functionally characterized by experiments

Methods

Results

Conclusion