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Ryegrass ( Lolium perenne  L.), a high-quality forage grass, is a good nutrient source for herbivorous livestock. However, improving nitrogen use efficiency and avoiding nitrate toxicity caused by excessive nitrogen are continual challenges in ryegrass production. The molecular mechanism underlying the response of ryegrass to nitrogen, especially excessive nitrogen, remains unclear. In this study, the transcriptomic changes under different nitrogen levels were investigated in perennial ryegrass by high-throughput next-generation RNA sequencing. Phenotypic characterization showed that treatment with half of the standard N concentration (N0.5) led to a better growth state than the other three treatments. The treatments with the standard N concentration (N1) and treatments with ten times higher than the standard N concentration (N10) contained excessive nitrogen, which placed stress on plant growth. Analysis of differentially expressed genes indicated that 345 and 104 genes are involved in the regulation of nitrogen utilization and excessive nitrogen stress, respectively. KEGG enrichment analysis suggested that “photosynthesis-antenna proteins” may respond positively to appropriate nitrogen conditions, whereas “steroid biosynthesis”, “carotenoid biosynthesis” and “C5-branched dibasic acid metabolism” were identified as the top significantly enriched pathways in response to excessive nitrogen. Additionally, 21 transcription factors (TFs) related to nitrogen utilization were classified into 10 families, especially the AP2-EREBP and MYB TF families. Four TFs related to excessive nitrogen stress were identified, including LOBs, NACs, AP2-EREBPs and HBs. The expression patterns of these selected genes were also analyzed. These results provide new insight into the regulatory mechanism of ryegrass in response to nitrogen utilization and excessive nitrogen stress.

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Transcriptome profiling revealed candidate genes, pathways and transcription factors related to nitrogen utilization and excessive nitrogen stress in perennial ryegrass