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 rtcs 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 rtcs 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 rtcs 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 rtcs 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.

SUMMARY Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan‐genome characterization. In this study, we generated a high‐quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long‐read sequencing and Hi‐C scaffolding, which yield highly contiguous, chromosome‐length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high‐quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: “missing a small part” in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis‐like cell death were detected in qk1 endosperm. Our study provides the community with a high‐quality genome sequence and a large collection of mutant germplasm.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant–pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.