Results

Cloning and functional prediction of the tobacco NtAN3 gene

Specific primers were designed based on the CDS sequence of the tobacco NtAN3 gene, and NtAN3 was subsequently amplified using PCR. Based on the physicochemical analysis of AtAN3 from Arabidopsis thaliana and tobacco NtAN3 proteins (Table 1), both proteins consist of 210 amino acids. NtAN3 has a molecular weight of 22491.79 Da and AtAN3 is 22463.62 Da. Their isoelectric points are 5.78 and 6.02 (both < 7), confirming that they are acidic proteins. The aliphatic indices are 54.90 and 55.38, reflecting a moderate level of aliphatic amino acids. The hydropathy indices are −0.80 and −0.72, and the instability indices are 71.50 and 67.13 for AtAN3 and NtAN3 correspondingly. These similar physicochemical properties suggest functional similarity between AtAN3 and NtAN3.

proteins | Number of amino acids | Molecular weight | Isoelectrc point | Aliphatic index | hydropathy index | Instability index
AtAN3 | 210.00 | 22463.62 | 5.78 | 54.90 | −0.80 | 71.50
NtAN3 | 210.00 | 22491.79 | 6.02 | 55.38 | −0.72 | 67.13

Analysis of functional domains (Supplementary Fig. 3) revealed that both proteins harbor the SSXT domain and share structural similarities. Purple bars denote conserved amino acid regions, whereas grey lines represent non-conserved polypeptide sequences. In addition, predicting and analyzing the secondary structures of AtAN3 and NtAN3 proteins(Table 2) showed that AtAN3 and NtAN3 the proportions of α-helix were 39.52% and 36.67%; β-turn accounted for 8.10% in both proteins; extended strand occupied 13.33% and 14.76%, while random coil made up 39.05% and 40.48%. These data demonstrate a high degree of similarity in the secondary structures between AtAN3 and NtAN3. Meanwhile, we used thethree-dimensional structural models of AtAN3 and NtAN3 proteins were predicted using the Phyre2 online server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)(Supplementary Fig. 4), their tertiary structures also exhibited strong resemblance, further supporting the high functional similarity of these two proteins.

Proteins | Alpha helix | Extended strand | Beta turn | Random coil
AtAN3 | 39.52 | 13.33 | 8.10 | 39.05
NtAN3 | 36.67 | 14.76 | 8.10 | 40.48

Effect of NtAN3 on tobacco leaf area

To investigate the effect of the tobacco NtAN3 gene on tobacco leaves, we constructed NtAN3 gene overexpression mutant plants (G27) and silencing mutant plants (M21) and measured the leaf length and width of wild-type and mutant plants. Based on the developmental results of tobacco mutant leaf length and width at different stages (Fig. 1), The leaf length of mutant plants G27 and M21 showed significant differences during the rosette stage, prosperous growing stage, flower budding stage, and central flower opening stage. But for leaf length, the significant difference was only detected at flower budding stage, between WT and G27. In contrast, there were significant differences in leaf width between G27 and WT, and M21 and WT during the flower budding stage. This indicates that NtAN3 promotes the growth of tobacco leaf length and width more during the flower budding stage. We then selected mutant plants in the flower budding stage for further analysis.

Fig. 1: Leaf length and leaf width of the mutant and wild type at different developmental times. Note: All data are means of three biological replicates. Error bars indicate standard error. Different lowercase letters indicate significant differences at P < 0.05. Wild type: WT; overexpression mutant plants: G27; silencing mutant plants: M21.

Sequencing analysis of mutant lines

Specific primers were designed to amplify the partial coding sequence (CDS) of endogenous tobacco NtAN3 for Sanger sequencing. PCR amplification was performed on positive mutant lines using primers designed flanking the target gene. The amplified products were digested with restriction enzymes, and the digested fragments were sent to a commercial sequencing company for sequence verification. Sequencing results are presented in Supplementary Fig. 1.

Analysis of target gene expression in mutant lines

Gene expression level in leaves serves as a direct indicator to quantify the abundance of the target gene in plant leaf tissues. Primers were designed based on the sequence of the target gene for quantitative real-time PCR (qRT-PCR) analysis of gene expression. Representative G27 and M21 were selected for expression quantification (Supplementary Fig. 2). Total RNA was extracted from tobacco leaf tissues using RNAiso Plus (Trizol) reagent following the protocols of the RNAprep Pure Plant Kit (TIANGEN, China). First-strand cDNA was synthesized with the FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, China) according to the manufacturer's instructions. All procedures were performed on ice to avoid RNA degradation. The results revealed significantly altered transcription levels of the target gene in both mutant genotypes. Specifically, the transcript abundance was markedly higher in the overexpression mutant and considerably lower in the silenced mutant relative to wild-type plants. These findings confirm that the genetic modification effectively regulated the transcription of the target gene.

Cytological analysis

To further explore the cytological basis of leaf morphological changes, paraffin sectioning and microscopic observation of leaf tissues were performed(Fig. 2, Supplementary Fig. 5). Quantitative analyses of blade thickness, cell number and cell size were conducted based on leaf paraffin cross-sections across WT, G27 and M21 tobacco leaf. All original measurement data and statistical outcomes are compiled in Supplementary Table 4. Cytological analysis showed significant differences in cell structure between wild-type and mutant plants. The results showed NtAN3 simultaneously regulates cell proliferation and cell expansion, and the elevation in cell number serves as the major contributor to leaf enlargement in overexpression plants.

Fig. 2: A:Blade thickness; B:Cell number; C:Cell size.D:Statistical chart.Note:Fig. 2C is a fixed-size statistical region cropped from panel Fig. 2B with equal magnification.

Transcriptome analysis of leaf area in tobacco

Using mutant plants G27 and M21 in the flower budding stage and their wild types as sequencing materials, 9 cDNA libraries were constructed and sequenced on the Illumina HiSeq Xten platform. A total of 116.54 Gb of clean data was obtained, with each sample generating more than 11.25 Gb of clean read sequences in RNA-Seq, which were then used for further analysis. Clean reads were mapped to the K326 genome, with approximately 95.70% to 96.09% of clean reads successfully mapped to the genome (Table 3). A total of 1697 differentially expressed genes were identified between WT vs G27, and 2388 differentially expressed genes were identified between WT vs M21 (Fig. 3A). This indicates that there are relatively few differentially expressed genes between G27 and WT, but relatively many differentially expressed genes between M21 and WT. Among these, 1035 genes were upregulated and 662 genes were downregulated between WT vs G27; 1313 genes were upregulated and 1075 genes were downregulated between WT vs M21. There were 301 up-regulated genes and 449 down-regulated genes in G27 vs M21(Fig. 3B). These results indicate that there are significant differences in gene expression between mutant plants and wild-type plants during leaf development.

Sample | Clean reads | Q20(20%) | Q30(%) | GC(%) | Contrast ratio(%)
G27_3 | 85450348 | 97.61 | 93.55 | 43.87 | 81870733(95.81%)
G27_2 | 81446076 | 97.75 | 93.77 | 44.00 | 78036769(95.81%)
G27_1 | 88298714 | 97.56 | 93.43 | 43.72 | 84499711(95.7%)
M21_3 | 87907540 | 97.84 | 93.97 | 43.39 | 84292419(95.89%)
M21_2 | 76256784 | 97.88 | 93.98 | 43.84 | 73022356(95.76%)
M21_1 | 83200302 | 97.77 | 93.83 | 43.81 | 79797041(95.91%)
WT_3 | 84103392 | 97.66 | 93.70 | 44.18 | 80779276(96.05%)
WT_2 | 113185342 | 97.80 | 93.91 | 43.94 | 108690411(96.03%)
WT_1 | 93567268 | 97.76 | 93.85 | 43.82 | 89904925(96.09%)

Fig. 3: Distribution of differentially expressed genes (DEGs) between mutant plants and wild-type plants. (A) Venn diagram of differentially expressed genes (DEGs). (B) DEGs expression changes.

We further screened two groups of differentially expressed genes with opposite expression patterns between G27 and M21: (1) genes significantly upregulated in overexpression mutant plants and downregulated in silenced mutant plants; (2) genes significantly downregulated in overexpression mutant plants and upregulated in silenced mutant plants.Only one genes were identified for the first gene set, while no genes met the screening thresholds for the second group. The expression patterns and functional annotations of this one candidate genes are listed in Supplementary Table 5.

Enrichment analysis of differences gene

GO enrichment analysis was performed on the differentially expressed genes between WT vs G27, and WT vs M21. Differentially expressed genes (DEGs) were screened with the threshold of P-value ≤ 0.05, and the DEGs were defined as the common genes obtained from the intersection of three biological replicates. We found that most of the differentially expressed genes were involved in biological processes. In the enrichment analysis of G27(Supplementary Table 1), the biological process with the highest number of genes was polysaccharide binding and terpenoid metabolic process, with 22 genes, while the biological process with the lowest number of genes was 5-epi-aristolochene synthase activity, with only 8 genes. The biological process with the highest enrichment level was glucan endo-1,3-beta-d-glucosidase activity. In the enrichment analysis of M21(Supplementary Table 2), the isoprenoid biosynthetic process had the highest number of genes, with 34 genes, while the phloem development had the lowest number of genes, with only 6 genes, but the development of the phloem had the highest degree of enrichment. According to the GO enrichment analysis of G27 and M21, the vast majority of genes involved in terpenoid synthesis and glycosidase activity were significantly enriched. Genes enriched in terpenoid synthesis and metabolic pathways displayed the most significant transcriptional variation between G27 and M21, which hints at a potential correlation between terpenoid metabolism and leaf development.

Metabolic pathway analysis was performed based on KEGG annotation. The G27 upregulated genes were annotated to 106 pathways, with the highest number of genes enriched in the plant-pathogen interaction pathway, while the sesquiterpene and triterpene biosynthesis pathways showed the highest enrichment levels (Fig. 4A). The M21 upregulated genes were annotated to 113 pathways. The MAPK signaling pathway - plant had the highest number of enriched genes, while the synthesis and degradation of ketone bodies had the highest enrichment level (Fig. 4B). In addition, the most KEGG annotation entries were related to metabolism. Among these differentially expressed genes, 15 were related to carbohydrate metabolism and 5 were related to cell size development. Based on the KEGG analysis, we further studied specific functional genes related to leaf development.

Fig. 4: KEGG enrichment analysis of differentially expressed genes between WT vs G27 and WT vs M21. (A) KEGG analysis of differentially expressed genes between WT and G27. (B) KEGG analysis of differentially expressed genes between WT and M21.

Differential expression of genes related to carbohydrate metabolism in mutant plants

Combined with functional enrichment analysis, we characterized differentially expressed genes associated with starch, sucrose and galactose metabolism to explore how NtAN3 affects carbohydrate physiological responses in tobacco leaves(Fig. 5). In the starch and sucrose metabolic pathways, a total of 13 genes were detected, including 4 genes annotated as Sucrose Synthase (SUS), 7 genes annotated as E3.1.2.4, 1 gene annotated as invertase (INV), and 1 gene annotated as phospho-β-glucosidase(bglB).

Fig. 5: Genes related to starch and sucrose metabolism and galactose metabolism are differentially expressed in wild-type and mutant plants.Note:Cell size:This figure shows the expression fold changes of differentially expressed transcripts annotated as homologs of RAPTOR, ABP1, ABC1,ABC1 and T92 genes based on transcriptome sequence analysis.

Overexpression of genes related to cell development during leaf development

In mutant plants, there is a significant enrichment of the GO category related to cell size. In cell size, Nitab4.5_0000564g0140, Nitab4.5_0006318g0030, Nitab4.5_0000047g0010, Nitab4.5_0010603g0020, and Nitab4.5_0002093g0100 showed high expression in G27 (Fig. 5), I've added the expression levels of these genes in G27,M21 and WT to Supplementary Table 7. As summarized in Supplementary Table 7, transcript abundance (average replicate TPM) of the five key candidate genes was substantially higher G27, whereas their expression was distinctly decreased in M21 when compared with wild-type tobacco, indicating that cell size is a key process influencing leaf area. The transcript Nitab4.5_0000047g0010 was annotated as RAPTOR, Nitab4.5_0002093g0100 as ABP1, Nitab4.5_0000564g0140 and Nitab4.5_0006318g0030 as ABCB1, and Nitab4.5_0010603g0020 as T92. All of these genes exhibited higher expression levels in G27 relative to M21. The annotation information corresponding to these genes has been added to Supplementary Fig. 6. Implying their potential roles in promoting cell growth.

RT-qPCR

The expression levels of DEGs were determined by qRT-PCR to verify the reliability of the RNA-seq data (Fig. 6). The results showed that six DEGs exhibited the same trend in both RNA-Seq and qRT-PCR results, indicating that the RNA-seq data were reliable.

Fig. 6: Real-time PCR validation of 6 DEGs.