Background: MicroRNAs (miRNAs) play crucial roles in the regulation of plant development and growth, but little information is available concerning their roles during grain development under different nitrogen (N) application levels. Our objective was to identify miRNAs related to the regulation of grain characteristics and the response to different N fertilizer conditions. Results: A total of 79 miRNAs (46 known and 33 novel miRNAs) were identified that showed significant differential expression during grain development under both high nitrogen (HN) and low nitrogen (LN) treatments. The miRNAs that were significantly upregulated early in grain development target genes involved mainly in cell differentiation, auxin-activated signaling, and transcription, which may be associated with grain size; miRNAs abundant in the middle and later stages target genes mainly involved in carbohydrate and nitrogen metabolism, transport, and kinase activity and may be associated with grain filling. Additionally, we identified 50 miRNAs (22 known and 28 novel miRNAs), of which 11, 9, and 39 were differentially expressed between the HN and LN libraries at 7, 17, and 27 days after anthesis (DAA). The miRNAs that were differentially expressed in response to nitrogen conditions target genes involved mainly in carbohydrate and nitrogen metabolism, the defense response, and transport as well as genes that encode ubiquitin ligase. Only one novel miRNA (PC-5p-2614_215) was significantly upregulated in response to LN treatment at all three stages, and 21 miRNAs showed significant differential expression between HN and LN conditions only at 27 DAA. We therefore propose a model for target gene regulation by miRNAs during grain development with N-responsive patterns. Conclusions: The potential targets of the identified miRNAs are related to various biological processes, such as carbohydrate/nitrogen metabolism, transcription, cellular differentiation, transport, and defense. Our results indicate that miRNA-mediated networks, via posttranscriptional regulation, play crucial roles in grain development and the N response, which determine wheat grain weight and quality. Our study provides useful information for future research of regulatory mechanisms that focus on improving grain yield and quality.

Storage protein distribution in wheat-grain endosperm is heterogeneous, but the underlying molecular mechanism remains unclear. Two parts of the endosperm region, the innermost endosperm (IE) region and the remaining endosperm (RE) region, grown under low nitrogen (LN) and high nitrogen (HN) treatments were used to perform metabolomic and transcriptomic analysis. We identified 533 and 503 differentially expressed genes (DEGs) with at least a two-fold expression change (p < 0.05) between IE and RE, among which 81 and 78 transcripts under LN and HN, respectively, related to carbon and nitrogen metabolism, and encoded transcription factors or proteins involved in post-translational modification (PTM). The significantly differentially abundant metabolites between IE and RE were mainly amino acids, N-compounds, carbohydrates, and nucleic acids. More upregulated transcripts and metabolites were identified in RE than IE under HN conditions, indicating that HN activates metabolism in the endosperm periphery. In addition to carbon and nitrogen metabolism, transcription factors and protein PTMs, such as phosphorylation and acetylation, might determine the protein heterogeneous distribution between IE and RE and its response to nitrogen fertilizer supply.