WGCNA analysis and identification of key genes in tobacco in response to different nitrogen levels.

BACKGROUND

Nitrogen (N) supply directly impacts growth and quality in flue-cured tobacco. To decipher molecular responses to N gradients, we integrated transcriptomics and weighted gene co-expression network analysis (WGCNA) on leaves from four N treatments: 0 (inherent soil fertility), 60 (low), 105 (standard), and 150 kg/hm (high).

RESULTS

Phenotypic analysis revealed dose-dependent increases in leaf nitrogen content with higher N application, accompanied by excessive vegetative growth and delayed maturity at 150 kg/hm. Transcriptome sequencing identified 47,216 genes, with differentially expressed genes (DEGs) increasing linearly with N levels (1,458-2,147 DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment highlighted nitrogen metabolism pathways, yielding 14 DEGs (11 in assimilation, 3 in transport). Weighted gene co-expression network analysis (WGCNA) uncovered two modules (lightcyan1 and black) strongly associated with N responses, harboring transcription factors NtERF11 (AP2/ERF), NtWRKY3 (WRKY), and NtSRM1 (MYB). Sub-network analysis within these modules identified five hub genes: NtGLN1-1, two uncharacterized genes, NtDFC, and NtGDSL. NtGDSL may enhance nitrogen use efficiency (NUE) through stress-responsive mechanisms, while NtDFC could integrate N signaling with developmental processes. These findings provide novel insights into N regulatory networks in flue-cured tobacco.

CONCLUSIONS

This study reveals the effects of nitrogen application rates on flue-cured tobacco growth and gene expression. By identifying key transcription factors and genes regulating nitrogen metabolism, it provides a theoretical basis for dissecting nitrogen regulatory mechanisms, optimizing fertilization strategies, and improving nitrogen use efficiency in tobacco production.