The NtAN3 gene plays a key role in increasing tobacco leaf area.

Tobacco (Nicotiana tabacum L.) is an important cash crop, and the size of its leaves significantly influences both yield and quality. However, previous research often focused on cultivation measures to enhance yield and quality, while the study of the structure and function of genes at the molecular level has been overlooked. We measured the leaf areas of NtAN3 overexpressing mutants, silenced mutants, and wild type. Compared with the wild type, the leaf area of the overexpressing mutant was significantly increased, and the leaf area of the silenced mutant was significantly decreased, indicating that NtAN3 plays a role in regulating the leaf area of tobacco. Cytological observations based on paraffin sections revealed that NtAN3 positively modulates leaf cell proliferation and expansion at the early developmental stage, thereby determining the final leaf area of tobacco. To further explore the downstream transcriptional changes triggered by altered NtAN3 expression, we performed comparative transcriptome sequencing using leaf samples collected at the flower budding stage. We selected two mutants, G27 and M21, which have significant differences in leaf area, and their parents for transcriptome analysis. There were 1697 differentially expressed genes in WT vs G27 and 2388 differentially expressed genes in WT vs M21. Functional enrichment showed remarkable transcriptional variations in carbohydrate metabolism pathways. Further analysis of starch, sucrose and galactose metabolism demonstrated that upregulated genes in these pathways mediate secondary metabolic adjustments. In addition, Nitab4.5_0000564g0140, Nitab4.5_0006318g0030, and Nitab4.5_0002093g0100 participate in late leaf metabolic responses induced by NtAN3. This transcriptomic research enriches our knowledge of the complete tobacco leaf regulatory network and provides molecular references for high-yield tobacco breeding.