Introduction

Tobacco (Nicotiana tabacum L.) is an economic crop harvested for its leaves. The size and shape of the leaves jointly determine the plant's effective photosynthetic area, which influences the yield and quality of photosynthetic products per leaf [1]. However, the dense tissue structure and narrow, thick leaves of upper tobacco leaves severely impair their yield and quality, resulting in low usability of upper tobacco leaves [2,3]. AN3 (ANGUSTIFOLIA3) is an important transcription co-activator in plants and plays a crucial role in leaf area development [4]. Research has shown that AN3 plays a key role in regulating cell proliferation and leaf primordia expansion. In Arabidopsis, overexpression of AN3 increases leaf width, leading to larger leaf area, while AN3 functional deficiency or mutation results in reduced cell numbers, causing narrower leaves and smaller leaf area [[4], [5], [6], [7]]. In recent years, transcriptomic studies have provided a molecular basis for leaf area development in Arabidopsis [4] and Chinese cabbage [8]. However, differences still exist in the expression patterns of AN3 gene and the interaction mechanisms of its upstream and downstream regulatory networks across different crops.

The growth and development of plant leaves is a complex process involving a large number of genes. Research shows that leaf morphology is influenced by a variety of genetic factors, such as plant hormones, transcription factors, and miRNAs [5,7,9]. In addition, there is a close relationship between leaf development and carbohydrate metabolism [10].Sucrose and starch metabolism are important components of carbohydrate metabolism [11]. Sucrose is ultimately broken down into hexoses or their derivatives, which are used in various metabolic and biosynthetic processes. Research has shown that sucrose synthase (SUS) and invertase (INV) in plants can catalyze the breakdown of sucrose [12,13]. INV not only participates in primary carbon metabolism but also plays a regulatory role in plant growth and development [14].

Cell size plays an important role in leaf development [15]. The target of rapamycin (TOR) pathway plays a crucial role in plant growth and development [16]. In addition, it has been reported that ABCB plays an important role in auxin transport [17].Auxin-binding protein 1 (ABP1) is an auxin receptor that is closely related to plant development [18].

In tobacco, leaf area is a key indicator of plant growth and development, yield formation, and quality regulation. Previous studies have shown that cultivation practices such as planting density, number of leaves retained, and fertilization can improve leaf area size [[19], [20], [21]]. Since the AN3 gene in Arabidopsis can regulate leaf area at the molecular level [22]. Therefore, this study preliminarily revealed the role of the AN3 gene in increasing tobacco leaf area and performed transcriptomic sequencing on mutant plants, followed by enrichment analysis of differentially expressed genes, providing new insights into the biological processes involving the synergistic effects of multiple genes in enlarging leaf area. These findings contribute to further elucidating the mechanisms underlying tobacco leaf development.