Single-cell transcriptome analyses reveal cellular and molecular responses to low nitrogen in burley tobacco leaves

Tobacco (Nicotiana tabacum) is cultivated and consumed worldwide. It requires great amounts of nitrogen (N) to achieve the best yield and quality. With a view to sustainable and environmentally friendly agriculture, developing new genotypes with high productivity under low N conditions is an important approach. It is unclear how genes in tobacco are expressed at the cellular level and the precise mechanisms by which cells respond to environmental stress, especially in the case of low N. Here, we characterized the transcriptomes in tobacco leaves grown in normal and low-N conditions by performing scRNA-seq. We identified 10 cell types with 17 transcriptionally distinct cell clusters with the assistance of marker genes and constructed the first single-cell atlas of tobacco leaves. Distinct gene expression patterns of cell clusters were observed under low-N conditions, and the mesophyll cells were the most important responsive cell type and displayed heterogene responses among its three subtypes. Pseudo-time trajectory analysis revealed low-N stress decelerates the differentiation towards mesophyll cells. In combination with scRNA-seq, WGCNA, and bulk RNA-seq results, we found that genes involved in porphyrin metabolism, nitrogen metabolism, carbon fixation, photosynthesis, and photosynthesis-antenna pathway play an essential role in response to low N. Moreover, we identified COL16, GATA24, MYB73, and GLK1 as key TFs in the regulation of N-responsive genes. Collectively, our findings are the first observation of the cellular and molecular responses of tobacco leaves under low N stress and lay the cornerstone for future tobacco scRNA-seq investigations.