Discussion

Research has demonstrated that the AN3 gene exerts a regulatory influence on leaf growth and development, as evidenced by studies on arabidopsis [5,7] and Chinese cabbage [8]. Transcriptome analysis has effectively revealed the molecular basis of leaf area growth and development in both arabidopsis [23] and Chinese cabbage [8]. However, the molecular basis of leaf area growth and development regulated by the AN3 gene in tobacco has not been reported in the extant literature. The development of upper leaves in tobacco is a critical factor in enhancing both yield and quality [2,3]. From this perspective, comparative transcriptomic analysis between wild-type and mutant plants was performed to characterize downstream transcriptional changes triggered by altered NtAN3 expression in leaves.

Carbohydrates are important factors in maintaining plant growth and development [24]. Carbohydrate metabolism regulates leaf growth and development by influencing key regulatory genes and enzymes [25]. Through KEGG enrichment analysis of upregulated genes in mutant plants, we found that genes related to carbohydrate synthesis were significantly enriched. To clarify the influences of NtAN3 on carbohydrate synthesis metabolism in tobacco leaves, we focused on starch and sucrose metabolism, as well as galactose metabolism.

Sucrose is one of the important forms of carbohydrate storage and transport in plants [26]. After being unloaded into storage tissues via the phloem, sucrose is converted into hexoses, which serve as carbon sources and energy for plant utilization [27]. This process is primarily catalyzed by SuS [11] and INV [28].In this study, 13 related genes were identified, which are involved in sucrose and starch metabolism, including 1 INV, 4 SUS, 7 E3.2.1.4, and 1 bgIB. G27 expression levels were higher than WT, and WT expression levels were higher than M21. INV and SUS promote the synthesis of d-fructose-6-phosphate and d-fructose [29], while E3.2.1.4 and bgIB promote d-glucose synthesis [30]. These monosaccharides provide energy for leaf development. This indicates that the overexpression of AN3 promotes the expression of enzymes involved in sucrose and starch metabolism, and generates secondary effects in tobacco leaves.

The intermediate products produced by galactose metabolism are key products in the synthesis of hemicellulose and pectin [31]. These components give the cell wall elasticity and mechanical strength, directly affecting the expansion capacity of leaf cells. INV genes are primarily involved in hexose accumulation and cell proliferation [32] regulating cell elongation through glucose-mediated auxin signaling [33]. In this study,two INV genes involved in the galactose metabolic pathway exhibited higher transcript abundance in G27 relative to M21, suggesting a potential correlation between NtAN3 accumulation and the expression of these INV genes.

Cell size is particularly important for plant growth and development [34]. Studies have reported that the rapamycin (TOR) kinase is a major glucose signaling mediator; it controls plant growth and development by integrating nutritional and energy signals, as well as growth factors, hormones, and environmental signals [35]. The kinase encoded by the TOR gene causes cells to enlarge, resulting in larger leaves [36]. Additionally, overexpression of the Arabidopsis ABP1 gene leads to cell expansion [37]. It has been reported that ABCB proteins are auxin transporters, and CsABCB19 regulates leaf structure by mediating auxin accumulation and transport [38,39]. Research indicates that cotyledon cell expansion depends on the ABCB19 protein to mediate the uptake of auxin in plants [40]. No reports have been made on the association between the T92 gene and leaf development. Transcriptomic analysis revealed altered expression of five genes associated with cell size regulation in mutant plants. However, cytological quantification showed no significant difference in cell size between overexpression and wild-type tobacco. We speculate that transcriptional changes of these genes cannot independently drive cell expansion due to functional redundancy or post-transcriptional regulatory compensation. The enlarged leaf area of NtAN3 overexpression lines is primarily determined by enhanced cell proliferation rather than cell expansion.

Notably, only one oppositely expressed gene was screened, which indicates that NtAN3 modulates leaf development via a small set of core downstream targets. Database annotation revealed that this candidate gene is involved in auxin-associated plant growth. We will perform further functional verification on this gene in subsequent experiments to elucidate the regulatory pathway of NtAN3.

Previous studies have confirmed that AN3 functions as a transcriptional co-activator without intrinsic MYB DNA-binding capacity. AN3 interacts with MYB-containing GRF transcription factors to co-regulate cell cycle-related genes, indirectly facilitating leaf cell proliferation through strengthening GRF transcriptional activity in Arabidopsis [23]. ABP1 forms a complex with the TMK kinase, acting as an extracellular auxin receptor to mediate rapid responses [41]. We reasonably speculate that AN3 overexpression may elevate ABP1 expression, further improve auxin response and consequently increase leaf area. According to transcriptome analysis, the gene annotated as an ABP1 homolog exhibited elevated expression in AN3-overexpressed plants (Fig. 5). We propose that the elevated ABP1 level enhances auxin response, which consequently increases leaf area. Results showed that the AN3 gene influences the expression of sugar metabolism enzymes(SUS_,_ INV_,_ blgB), whose increased activity raises intracellular glucose concentration, activating the TOR signaling pathway. Studies indicate that the P-glycoprotein encoded by ABCB1 is involved in the transmembrane transport of auxin [42]. Therefore, we tentatively propose that AN3 is involved in the regulation of tobacco leaf area through a putative multi-level network consisting of transcription factors, transport proteins and metabolic enzymes.

In this study, there are still several limitations that need to be acknowledged. Firstly, these mutant materials were preliminarily screened and confirmed by phenotypic traits and basic molecular identification only. Further quantitative verification at the transcriptional level is still required. In future research, we will supplement relevant gene expression detection and collect more homologous gene sequences to optimize relevant experimental analysis. Secondly, transcriptome analysis mainly reflects dynamic changes in gene expression and downstream molecular responses during leaf development. The significant enrichment of carbohydrate metabolism pathways indicates that these metabolic processes are closely correlated with leaf growth. At this stage, we cannot fully confirm whether the alteration of carbohydrate metabolism serves as an upstream causal factor initiating leaf area expansion, or is a secondary metabolic response resulting from changes in leaf developmental status. Further genetic and physiological experiments are required to clarify their definite causal relationship in future research. It should be noted that carbohydrate metabolism, terpenoid biosynthesis and MAPK signaling pathways are widely involved in multiple plant growth and metabolic processes, and are not uniquely specific to leaf area development. In this study, we only performed general pathway enrichment analysis, and did not further screen and prioritize core hub genes within these pathways. Further screening of key regulatory factors is required to clarify their specific functions in leaf development.