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/hm² (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/hm². 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. Overall design: The experiment was conducted with the same phosphorus and potassium fertilization levels (phosphorus 105 kg/hm2, potassium 210 kg/hm2), employing four nitrogen fertilizer levels: no nitrogen fertilizer, 0 kg/hm2 (T1); nitrogen deficiency, 60 kg/hm2 (T2); standard application, 105 kg/hm2 (T3); and nitrogen excess, 150 kg/hm2 (T4).The experiment utilized a randomized complete block design with plot size of 66.7 m2, replicated three times.Standard float bed seedling raising method was employed, selecting vigorous tobacco seedlings, transplanted on April 28, 2022.Planting density was 16,492 plants/hm2 with row spacing of 1.10 m and plant spacing of 0.55 m, equivalent to 1100 plants/acre, with protective rows around.Only healthy and uniform four-leaf stage tobacco seedlings free from pests and diseases were selected for transplantation.For T3 treatment, fertilization ratio was N:P2O5= 1:1:2, with nitrogen provided as urea (Red Label, N = 46.2%), phosphorus as calcium superphosphate (Red Star, P2O5 = 16.0%), and potassium as potassium sulfate (Rubobuo, K2O = 52.0%).Potassium fertilizer was applied at 210 kg/hm2, with nitrogen and potassium split between basal (70%) and topdressing (30%) applications. Basal fertilizer was applied before transplanting, and topdressing during the topping stage; all phosphorus fertilizer was used as basal at a rate of 105 kg/hm2.