Conclusions

This study clarified the role of the AN3 gene in tobacco by measuring the leaf area of mutant plants. Transcriptome analysis of mutant plants and wild-type plants showed that the expression level of the AN3 gene affects overall gene expression. There were 2388 differentially expressed genes between M21 and WT, and 1697 differentially expressed genes between G27 and WT. KEGG enrichment analysis of these differentially expressed genes revealed that carbohydrate metabolism pathways and cell size are associated with leaf development. We focused on genes related to sucrose and starch metabolism, galactose metabolism, and cell size, and found that the overexpression of these genes may be associated with increased leaf area. We hypothesize that NtAN3 may positively modulate the expression of ABCB, ABP1, INV, SUS and bgIB potentially through the TOR signaling pathway, forming a tentative regulatory cascade linking signal perception to metabolic reprogramming to accelerate leaf cell proliferation and expansion. However, since we have not directly measured TOR activity, endogenous auxin concentrations or downstream signaling outputs in this work, this proposed regulatory network remains a hypothetical framework that requires comprehensive experimental verification in follow-up research. In summary, this study provides new insights into the mechanism by which the AN3 gene increases tobacco leaf area. However, further studies are needed to elucidate the developmental mechanism of tobacco leaf area by investigating the synergistic effects of the AN3 gene with these genes.