Comparative RNA-Seq Analysis Reveals That Regulatory Network of Maize Root Development Controls the Expression of Genes in Response to N Stress

<div><p>Nitrogen (N) is an essential nutrient for plants, and it directly affects grain yield and protein content in cereal crops. Plant root systems are not only critical for anchorage in the soil, but also for N acquisition. Therefore, genes controlling root development might also affect N uptake by plants. In this study, the responses of nitrogen on root architecture of mutant <i>rtcs</i> and wild-type of maize were investigated by morphological and physiological analysis. Subsequently, we performed a comparative RNA-Seq analysis to compare gene expression profiles between mutant <i>rtcs</i> roots and wild-type roots under different N conditions. We identified 786 co-modulated differentially expressed genes (DEGs) related to root development. These genes participated in various metabolic processes. A co-expression cluster analysis and a cis-regulatory motifs analysis revealed the importance of the AP2-EREBP transcription factor family in the rtcs-dependent regulatory network. Some genotype-specific DEGs contained at least one LBD motif in their promoter region. Further analyses of the differences in gene transcript levels between <i>rtcs</i> and wild-type under different N conditions revealed 403 co-modulated DEGs with distinct functions. A comparative analysis revealed that the regulatory network controlling root development also controlled gene expression in response to N-deficiency. Several AP2-EREBP family members involved in multiple hormone signaling pathways were among the DEGs. These transcription factors might play important roles in the rtcs-dependent regulatory network related to root development and the N-deficiency response. Genes encoding the nitrate transporters NRT2-1, NAR2.1, NAR2.2, and NAR2.3 showed much higher transcript levels in <i>rtcs</i> than in wild-type under normal-N conditions. This result indicated that the LBD gene family mainly functions as transcriptional repressors, as noted in other studies. In summary, using a comparative RNA-Seq-based approach, we identified DEGs related to root development that also participated in the N-deficiency response in maize. These findings will increase our understanding of the molecular regulatory networks controlling root development and N-stress responses.</p></div

Gene annotation of co-modulated DEGs, analyzed and visualized by MapMan.

<p>Each block represents average expression changes for one gene. Three rows of blocks are shown to demonstrate wild-type-specific N-deficiency DEGs, <i>rtcs</i>-specific N-deficiency DEGs and rtcs-dependent common DGEs from bottom to top.</p

RNA-Seq analysis of <i>rtcs</i> and wild-type roots transcriptome under normal-N condition.

<p>(A) Venn diagram analysis of DEGs between wild-type and <i>rtcs</i> at four developmental time points. (B) Co-modulated differential expression transcription factors (TFs) between wild-type and <i>rtcs</i> at four time points. TFs were grouped by family and the number of differential expression TFs is indicated. Each column represents average expression differences across the TF family at one time point. (C) Clustering co-modulated DEGs based on the expression profiles (FPKM values were log10-transformed). The top GO terms and corresponding DEGs number are shown on the right side, “*” represents significant enrichment (FDR <0.05).</p

Gene annotation and functional enrichment analysis for DEGs in response to N-deficiency.

<p>(A) Cross-comparison of pathway enrichment analysis among DEGs in response to N-deficiency. The <i>y</i>-axis corresponds to the KEGG pathway, and the <i>x</i>-axis shows the enrichment factor. The color of the dot represents the <i>q</i>-value, and the size of the dot represents the number of DEGs mapped to the reference pathways. (B) Cross-comparison of enriched GO terms among DEGs in response to N-deficiency. The top GO terms and corresponding DEGs number are shown on the right side, “*”represents significant enrichment (FDR <0.05).</p

Co-modulated DEGs associtated with root development and response to N stress were involved in many levels of biological regulation.

<p>Co-modulated DEGs associtated with root development and response to N stress were involved in many levels of biological regulation.</p