Methods

Obtaining plant material

In this research, the tobacco strain NC82 was utilized, sourced from the Guizhou Provincial Key Laboratory for Tobacco Quality Improvement and Efficiency Enhancement at Guizhou University. This tobacco leaf is of the small-leaf type, with narrow and small upper leaves, offering excellent quality and high stability. Mutant samples were generated at the Guizhou Provincial Key Laboratory for Tobacco Quality Improvement and Efficiency Enhancement, while greenhouse field tests were undertaken at the Yangwu Tobacco Research Base affiliated with Guizhou University. The genetic transformation was introduced into the leaves of the NC82 tobacco strain using an Agrobacterium-mediated approach. To generate silenced mutants, an RNAi vector targeting NtAN3 was constructed. A specific fragment of the NtAN3 CDS was amplified by PCR and inserted into the pFGC5941 vector in sense and antisense orientations. The recombinant construct was transformed into Agrobacterium tumefaciens GV3101. Genetic transformation of tobacco cultivar NC82 was conducted via the Agrobacterium-mediated leaf disc method. Regenerated resistant seedlings were screened on selective medium, and RT-qPCR was used to identify transgenic lines with significantly suppressed NtAN3 expression.We ensured that the collection of plant material andexperimental research and field studies on plants compliedwith relevant institutional, national, and internationalguidelines and legislation.

Field experimental design

The field experiment employed a randomized complete block design with three replications. Each plot consisted of four rows, with 15 plants per row, at a spacing of 110 cm × 55 cm. Border rows were established around the experimental area, and the first and last plant in each row were excluded from sampling. Fertilization, plant density, and other crop management practices followed the high-quality tobacco production protocol of the research station.

Agronomic trait measurement

Leaf length and leaf width at the 16th, 17th, and 18th leaf positions were recorded for both mutant and wild-type plants at each developmental stage. We classified plant developmental stages according to morphological characteristics, and the classification criteria were defined as follows: Rosette stage: Plants reach the rosette standard with 12–13 expanded leaves. The ratio of plant width (horizontal growth) to plant height (vertical growth) is approximately 2:1, and the plant shape resembles a hemisphere. Prosperous growing stage: The period spanning from the rosette stage to flower budding stage. Flower buding stage: The stage when flower buds are fully exposed. Central flowers opening stage: The stage when the first central flower of the plant opens. Meanwhile, leaf samples were collected and preserved under two conditions: frozen at appropriate temperatures (frozen samples), and fixed in FAA (formalin-acetic acid-alcohol) solution (FAA-fixed samples) for further analysis.

Statistical analysis and reproducibility

ANOVA followed by Fisher's Least Significant Difference (LSD) post hoc test for multiple comparisons. A P-value < 0.05 was considered statistically significant. The exact P-values are indicated in the figures or figure legends where applicable.

RNA extraction and transcriptome sequencing

At the flower budding stage, three representative plants per line were selected. Leaves from the same position were collected, frozen at −80 °C, and then pooled per line before being sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for total RNA extraction (three replicates per treatment), cDNA library construction, and sequencing. Subsequent DEG screening was accomplished via Majorbio online platform with cutoff criteria: |log2(fold change)| ≥ 1 and q-value < 0.05.

Total RNA extraction and cDNA first strand synthesis

Total RNA from the leaves was isolated employing the RNAiso Plus TriZol technique. Subsequently, cDNA was generated following the instructions of the FastKing gDNA Dispelling RT SuperMix (TIANGEN) Reverse Transcription Kit.

Cloning and bioinformatics analysis of the NtAN3 gene

Primers tgtF and tgtR were designed using Primer6.0 software and synthesized by Qingke Biotechnology (Chongqing) Co., Ltd. The PCR output was inserted into the pBWA(V)HS-AN3 vector. Positive colonies were pinpointed using PCR, and the rDNAG1 and rDNAtlt3 products underwent enzymatic digestion followed by sequencing. A similarity comparison between NtAN3 and related genes was undertaken through NCBI. The physicochemical attributes, including hydrophilicity and hydrophobicity, of Arabidopsis thaliana AN3 and the acquired glycosidic acid sequences from tobacco homologs were scrutinized with the ProtParam tool.

Genetic transformation and characterization of NtAN3

The genetic transformation was executed using Agrobacterium infiltration. From this process, 30 overexpression plants and 6 silenced plants were identified. Following PCR amplification, 24 positive mutant plants were distinguished, comprising 20 overexpression mutants and 4 silenced mutants. For each selected transgenic line, three individual plantlets were taken as biological replicates to perform subsequent physiological and molecular measurements.

Sequencing verification of mutant lines

Genomic DNA was extracted from leaf tissues of mutant plants for PCR amplification. Gene-specific primers were designed to amplify the target fragment of the NtAN3 gene.AN3-F: GAGCCAGAACTCAGGGAAAC.AN3-R:ATCAGCAATAGCAGCAAGGT.

Histological observation and paraffin section analysis

To investigate the cytological differences in leaf structure among wild-type, G27 and M21, mature and fully expanded functional leaves at the same developmental stage(flower building stage) were selected for paraffin section preparation. Leaf tissues were cleaned and dried, and rectangular segments (2 mm × 5 mm) were cut from the middle area of the leaf, avoiding the main vein, to ensure consistent sampling positions.

The collected samples were immediately fixed in FAA solution (50% ethanol, glacial acetic acid, and formaldehyde at a volume ratio of 18:1:1) with a sample-to-fixative ratio of 1:20. Vacuum pumping was applied to remove internal air bubbles and promote full infiltration. Samples were fixed for more than 48 h. After fixation, tissues were rinsed three times with 70% ethanol for 2 h each and immersed in 70% ethanol overnight.

For dehydration, samples were treated with a gradient ethanol series: 70%, 80%, 95%, and two changes of absolute ethanol, with 2 h incubation for each step. Subsequently, tissues were cleared through a gradual xylene-ethanol gradient (1/3 xylene + 2/3 ethanol, 1/2 xylene + 1/2 ethanol, 2/3 xylene + 1/3 ethanol) and two pure xylene treatments. Safranin staining was performed during the clearing process to facilitate tissue localization.

For paraffin infiltration, tissues were incubated in a 1:1 mixture of xylene and paraffin, followed by two rounds of pure paraffin infiltration at 55–60 °C for approximately 3 h each. Paraffin with melting points of 52–54 °C or 56–58 °C was selected according to ambient temperature conditions.

Samples were embedded, trimmed into regular trapezoid blocks, and fixed on paraffin holders. Serial sections were first trimmed at 15–20 μm, and formal histological sections were prepared at a thickness of 8–12 μm. Sections were mounted on glass slides using Haupt's adhesive solution, unfolded in a 36 °C water bath, and dried at 36 °C for more than 24 h.

For staining, slides were deparaffinized and rehydrated through a xylene and ethanol gradient, stained with safranin and fast green, and then dehydrated and cleared gradually. Finally, specimens were mounted with neutral balsam and dried at 36 °C to prepare permanent sections.

All sections were observed and photographed using an Olympus light microscope. Leaf thickness measurements were performed on paraffin cross-section images captured under a 20× microscope objective. For mesophyll cell number counting and single cell size quantification, micrographs were acquired at a uniform 40× objective magnification. A fixed frame with identical pixel dimensions was used to delineate the statistical region on each 40× image, and all cells inside the equal-area frame were counted manually; meanwhile, the size of individual cells within the framed area was measured. Uniform magnification and standardized statistical frames ensured comparable data across wild-type and mutant plants.

Real-time fluorescence quantitative analysis of the NtAN3 gene

Total RNA was isolated from tobacco leaves of overexpressed, silenced, and wild type. The concentration and purity of the retrieved RNA were assessed using Nanodrop2000 and further validated through gel electrophoresis. A 1291-bp fragment of NtAN3 was amplified for gene cloning, and a separate short amplicon suitable for real-time PCR (100–150 bp) was used for expression quantification. The sequences of the synthesized primers are shown in Supplementary Table 6.Fluorescence experiments were conducted in line with the Talent qPCR PreMix (SYBR Green) kit's guidelines. The NtAN3 relative expression level was gauged using a fluorescence quantitative PCR machine. Gene expression was quantified employing the 2–ΔΔCt technique [43]. The Actin gene functioned as the reference gene, and each gene's evaluation was repeated thrice. qRT-PCR specific primers targeting the candidate genes were formulated using Primer6.0 and Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) available on NCBI. DynaPro Bio (Chongqing) Co. The fluorescence quantitative PCR system included 2×Talentq PCR PreMix 10 μL, 10 μM PrimerF 0.6 μL, 10 μM PrimerF 0.6 μL, cDNA 2 μL, and RNase-Free ddH2O to make final volume 20 μL. The PCR reaction settings were: an initial phase at 94 °C for 3 min, followed by 40 cycles each at 95 °C for 5 s, 50–60 °C for 10 s, and 72 °C for 15 s.