Identification, evolution, and expression analysis of the PEBP gene family in tobacco (Nicotiana tabacum) and its role in flowering regulation via interaction with 14-3-3 proteins

Abstract

The phosphatidylethanolamine-binding protein (PEBP) gene family plays a crucial role in regulating key biological processes in plants, such as flowering time, morphological architecture, and seed germination. However, the diversity, expression patterns, and functions of PEBP genes in Nicotiana tabacum remain largely unexplored. In this study, 32 PEBP gene family members were identified in Nicotiana tabacum and systematically classified into four subfamilies: FT-like (17 members), TFL1-like (9 members), MFT-like (2 members), and YbhB-like (4 members). Conserved motifs DPDxP and GxHR, essential for anion-binding activity, were identified in NtPEBPs. Promoter cis-acting element analysis indicated that NtPEBPs are extensively involved in light response, hormone regulation, stress response, and growth and development in tobacco. Transcriptome data revealed that some genes exhibit tissue-specific expression patterns, with certain NtBFT and NtTSF members showing high expression levels in leaves and flowers, while some members of the MFT-like and YbhB-like subfamilies were significantly expressed in dry capsules. Subcellular localization confirmed that NtATC4, NtBFT1, and NtBFT4 are localized in the cytoplasm, while NtFT7 is localized in both the nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays further demonstrated that NtATC4, NtFT7, NtBFT1, and NtBFT4 can all interact with the Nt14-3-3 protein, suggesting their potential involvement in the tobacco flowering process through the formation of flowering regulatory complexes. Virus-induced gene silencing (VIGS) assays revealed that silencing of NtATC4 delays flowering and promotes vegetative growth. In NtATC4-silenced plants, the expression of flowering-related genes NbAP1 and NbSVP was upregulated, whereas NbSOC1 expression was downregulated. This study provides a comprehensive gene inventory and interaction dataset for further research on NtPEBPs and identifies candidate targets for molecular breeding in tobacco.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08399-9.

Background

Nicotiana tabacum L., an important agricultural crop within the Solanaceae family, serves as a model system for molecular biology research and as a bioreactor for vaccine and pharmaceutical production [1, 2]. Like other crops, tobacco’s architectural morphology is shaped by both endogenous factors (e.g., hormonal signals, inter-organ nutrient competition) and exogenous factors (e.g., light, temperature, soil moisture, and nutrient availability) [3]. Furthermore, flowering time has a decisive impact on crop yield and morphological architecture [4, 5].

Phosphatidylethanolamine-Binding Proteins (PEBP) are a class of evolutionarily conserved proteins widely found in plants, animals, and microorganisms [6–8]. These proteins were initially isolated from bovine brain and named for their strong affinity for phosphatidylethanolamine [9, 10]. Structural analyses of several identified PEBP-like family members reveal that PEBPs possess a conserved domain featuring a large central β-sheet. This large β-sheet is formed in conjunction with a smaller β-sheet and an α-helix [11, 12]. In plants, the PEBP gene family regulates diverse biological processes. Some studies have found that PEBP genes not only regulate flowering time but also influence plant architecture [13]. Furthermore, PEBP genes are involved in regulating seed germination and dormancy [14–16]. In the model plant Arabidopsis thaliana, the PEBP gene family comprises seven members: FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), TERMINAL FLOWER 1 (TFL1), BROTHER OF FT AND TFL1 (BFT), MOTHER OF FT AND TFL1 (MFT), Arabidopsis thaliana CENTRORADIALIS (ATC) [17, 18], and AT5G01300 (PYBHB) [19]. These genes are classified into four subfamilies: FT-like, TFL1-like, MFT-like [20], and YbhB-like [21]. In Arabidopsis, FT, TSF, and MFT promote flowering, whereas TFL1, ATC, and BFT suppress it [22–25]. FT and TSF belong to the FT-like subfamily. Within the floral meristem, FT interacts with the bZIP transcription factor FD and 14-3-3 proteins [26, 27] to activate the expression of floral identity genes, including LEAFY and APETALA1 (AP1), thereby initiating floral meristem development [28, 29]. TFL1, ATC, and BFT belong to the TFL1-like subfamily. TFL1 regulates developmental transitions and inflorescence architecture by repressing the expression of floral identity genes. Loss of TFL1 function accelerates flowering time and inhibits the development of indeterminate inflorescences [30]. FT and TFL1 exhibit antagonistic functions in regulating flowering time [31]. Their coordinated regulation not only affects plant architecture and life cycle but also plays a crucial role in determining the timing of flowering [32]. MFT belongs to the MFT-like subfamily and is considered the common ancestor of both FT and TFL1 [33]. MFT acts as a flowering promoter, similar to FT and TSF, and its overexpression leads to early flowering [34]. Beyond influencing flowering time, MFT also controls seed germination by regulating abscisic acid (ABA) signaling through a negative feedback loop [14]. Additionally, MFT is involved in the crosstalk between ABA and brassinosteroid (BR) signaling during seed germination [14].

In Solanaceae crops, the PEBP family predominantly regulates flowering and plant architecture through antagonistic interactions between FT-like and TFL1-like subfamily members. In tomato, SFT (FT-like) promotes flowering and meristem activity, while SP (TFL1-like) antagonizes SFT to maintain vegetative growth; loss-of-function sp mutants exhibit determinate growth with synchronized fruit ripening, ideally suited for mechanical harvesting [35–40]. Analogously, CaFT-LIKE in pepper also functions as a floral promoter [41]. While potato and petunia possess 15 and 13 PEBP members respectively [42, 43], tobacco harbors at least 16 members including suppressors (NtFT1, NtFT2 and NtFT3) and promoters (NtFT4 and NtFT5) [44–46], yet lacks systematic characterization, suggesting a potentially more complex regulatory network in this allotetraploid species.

Although multiple members of the tobacco PEBP gene family have been identified and studied, a systematic bioinformatic analysis has not yet been conducted. This study employs bioinformatic techniques to identify 32 PEBP gene family members and analyzes their phylogenetic relationships, gene structures, protein motifs, and expression profiles across different tissues. These findings may provide valuable information for the classification of NtPEBP genes and lay the foundation for exploring the molecular mechanisms by which PEBP genes regulate plant architecture and flowering in tobacco.

Materials and methods

Plant materials and growth conditions

The cultivated common tobacco variety N. tabacum K326 and N. benthamiana were used as experimental materials. Seeds were sown in a substrate mixture of perlite, vermiculite, and peat soil at a ratio of 1:1:3, and cultivated in a constant-temperature growth chamber at 25 °C under a 16/8-hour light/dark cycle. All experimental materials were collected and frozen using liquid nitrogen, then stored at -70 °C until subsequent use.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from different plant materials using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). Genomic DNA was removed, and cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). The resulting cDNA was stored at − 80 °C.

Identification of PEBP family members in tobacco

The genomic sequences of N. tabacum, N. benthamiana, N. sylvestris and N. tomentosiformis were downloaded from the Sol Genomics Network (SGN) and zenodo database (https://solgenomics.net/ftp/genomes/Nicotiana_tabacum/edwards_et_al_2017/; https://solgenomics.net/ftp/genomes/Nicotiana_benthamiana/V261/; https://zenodo.org/records/8256252; https://zenodo.org/records/8256254) for the identification and subsequent analysis of the PEBP gene family members in tobacco [47, 48]. The conserved domain of PEBP (PF01161) was obtained from the Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam/PF01161/) [49]. HMMER 3.0 (http://hmmer.org/) was used to search the tobacco protein datasets and retrieve potential members of the tobacco PEBP family. The obtained genes were then validated using the InterPro database (https://www.ebi.ac.uk/interpro/), and genes lacking the complete conserved PEBP domain were excluded [50]. The physicochemical properties of NtPEBP proteins, such as molecular weight (kDa), theoretical isoelectric point, and instability index, were predicted using the ExPASy online tool (https://web.expasy.org/protparam/), while the subcellular localization of NtPEBP proteins was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) [51, 52].

Multiple sequence alignment and phylogenetic analysis

The PEBP family members in N. benthamiana, N. sylvestris and N. tomentosiformis were identified using the same methodology as that applied for N. tabacum. Multiple sequence alignment of PEBP protein sequences from N. tabacum, N. benthamiana, N. sylvestris, N. tomentosiformis, Arabidopsis thaliana, tomato, and pepper was performed using ClustalW [53]. A phylogenetic tree was constructed with IQ-TREE 2 based on the maximum likelihood method [54]. The Jones-Taylor-Thornton (JTT) model was selected as the amino acid substitution model for phylogenetic inference, with 1000 bootstrap replicates and all other parameters set to default. Finally, the phylogenetic tree was visualized and refined using Evolview 3.0 (https://www.evolgenius.info/evolview/) [55].

Nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) analysis

The CDSs of HKT genes were aligned by ClustalW 2.0based on the protein sequences. The NtPEBP gene duplication was confirmed based on two criteria: (a) the length of the shorter aligned sequence covered > 70% of the longer sequence; and (b) the similarity of the two aligned sequences were > 70% [56, 57]. Two genes separated by five or fewer genes in 100-kb chromosome fragment were considered as tandem duplicated genes. The Ka/Ks ratios for the identified duplicate gene pairs were subsequently calculated using Tbtools-II to assess the selection pressure acting upon them [58].

Genetic structure and conserved motif analysis

The Gene Structure Display Server (GSDS 2.0) (https://gsds.gao-lab.org/index.php) was used to generate schematic diagrams of gene structures [59]. The conserved motifs of the NtPEBP gene family members were predicted using the Multiple Em for Motif Elicitation (MEME) suite (https://meme-suite.org/meme/tools/meme) [60]. The number of predicted conserved motifs was set to 10, with all other parameters maintained at their default values. Finally, visualization was performed using Tbtools-II.

Analysis of cis-acting elements in the NtPEBP gene family

The 1500-bp 5’ sequences upstream of the NtPEBP genes were extracted as putative promoter regions. Potential cis-acting elements within these promoter regions were predicted using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [61]. Finally, the prediction results were analyzed, categorized, and visualized.

Prediction of the three-dimensional structure of the NtPEBP protein

The three-dimensional structures of NtPEBP proteins were predicted using the AlphaFold Server (https://alphafoldserver.com/) [62]. AlphaFold Server is an online service that generates high-accuracy biomolecular structure predictions—including proteins, DNA, RNA, ligands, and ions—on a unified platform, and supports modeling of chemical modifications for proteins and nucleic acids. The server is powered by the latest AlphaFold 3 model. During the prediction process, five initial models were generated, and the model with the highest predicted confidence (highest pLDDT score) was selected for further analysis.

Gene Expression Profile Analysis of NtPEBPs

RNA sequencing (RNA-seq) data were downloaded from the NCBI Sequence Read Archive with accession number SRP029183. The root, stem, and leaf materials in the data were obtained from four-month-old tobacco plants [63]. Clean reads obtained from the raw data were mapped to the N. tabacum genome using HISAT (version Nitab4.5) with default parameters [64]. The expression level of each gene was quantified as Transcripts Per Million (TPM) values, which were obtained using StringTie with default parameters [65, 66].

Part gene member expression level was further detected Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene-specific primers were provided in Table S1. Materials from different tissues were collected from tobacco plants at the fruiting stage. qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a 20 µL reaction volume. Samples from different plant materials were analyzed with three biological replicates using a Bio-Rad IQ5 Real-Time PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA). The amplification protocol consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. The relative gene expression levels were calculated using the 2^−ΔΔCt method.

Subcellular localization of NtPEBP protein

The genes NtATC4, NtBFT1, NtBFT4, and NtFT7 were cloned into the pCAMBIA1300-35 S-GFP vector and transformed into Agrobacterium tumefaciens strain GV3101. Primers were designed based on the sequences of NtATC4, NtBFT1, NtBFT4, and NtFT7 (Table S1). Agrobacterium suspensions harboring the recombinant constructs–control GFP, NtATC4-GFP, NtBFT1-GFP, NtBFT4-GFP, and NtFT7-GFP–were infiltrated into tobacco leaves. The transiently transformed tobacco leaves were cultured in darkness for 2–3 days, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) (Genview). Finally, protein localization was observed under a fluorescence microscope.

Yeast two-hybrid experiment

The coding sequences of NtATC4, NtBFT1, NtBFT4, NtFT7, and Nt14-3-3 genes were amplified using the designed primers (Table S1). NtATC4, NtBFT1, NtBFT4, and NtFT7 were cloned into the EcoR I and BamH I sites of the pGBKT7 vector. Nt14-3-3 was cloned into the EcoR I and BamH I sites of the pGADT7 vector. Yeast recipient cells co-transformed with the following plasmid combinations—pGADT7-Nt14-3-3 + pGBKT7-NtATC4, pGADT7-Nt14-3-3 + pGBKT7-NtBFT1, pGADT7-Nt14-3-3 + pGBKT7-NtBFT4, pGADT7-Nt14-3-3 + pGBKT7-NtFT7, pGADT7 + pGBKT7, pGADT7-Nt14-3-3 + pGBKT7, pGADT7 + pGBKT7-NtATC4, pGADT7 + pGBKT7-NtBFT1, pGADT7 + pGBKT7-NtBFT4, pGADT7 + pGBKT7-NtFT7, pGADT7-T + pGBKT7-53, and pGADT7-T + pGBKT7-Lam—were plated on double-dropout (SD) medium lacking the appropriate amino acids and incubated inverted at 30 °C for 2–3 days. Colonies larger than 2 mm in diameter were then transferred to quadruple-dropout SD medium and incubated inverted at 30 °C for 4–5 days.

Bimolecular fluorescence complementation assay

For the bimolecular fluorescence complementation (BiFC) assay, the coding sequences of NtATC4, NtBFT1, NtBFT4, NtFT7, and Nt14-3-3 were cloned into the vectors pXY-104-YFP-C (using KpnI and SalI sites) and pXY-106-YFP-N (using BamHI and SalI sites), with primers listed in Table S1. All constructs were subsequently transformed into Agrobacterium tumefaciens strain GV3101. Bacterial cultures harboring different plasmids were then mixed in equal volumes for co-infiltration into Nicotiana benthamiana leaves. This included five combinations: pXY-104-YFP-C with pXY-106-YFP-N as a negative control, and four experimental pairs consisting of pXY-104-NtATC4-YFP-C, pXY-104-NtBFT1-YFP-C, pXY-104-NtBFT4-YFP-C, or pXY-104-NtFT7-YFP-C, each co-infiltrated with pXY-106-YFP-N-Nt14-3-3. YFP fluorescence was observed under a fluorescence microscope at 48 h post-infiltration.

Virus-induced gene silencing of NtATC4

To investigate the role of NtATC4 in tobacco, Tobacco Rattle Virus (TRV)-based Virus-Induced Gene Silencing (VIGS) was performed using the pTRV1 and pTRV2 vectors. A gene-specific fragment of NtATC4 was amplified using designed primers (Table S1) and cloned into the BamHI and EcoRI sites of the pTRV2 vector. The resulting pTRV2-NtATC4 construct was then transformed into Agrobacterium tumefaciens strain GV3101. The empty pTRV2 vector served as a negative control, while pTRV2-PDS was used as a positive control. Bacterial cultures carrying pTRV1 and pTRV2 derivatives (OD₆₀₀ = 0.8) were mixed in a 1:1 ratio and infiltrated into the abaxial side of leaves of 4-week-old tobacco plants using a syringe. After 1–2 days of dark incubation, the plants were cultivated at 22 °C under a 16-hour light/8-hour dark photoperiod. Samples were collected three weeks after treatment for subsequent experiments. Three independent biological replicates were performed, with a minimum of ten plants per replicate for each treatment: pTRV2-NtATC4, the empty vector control, and the PDS control.

Statistical analysis

Perform statistical analysis and data visualization using the software GraphPad Prism (https://www.graphpad.com/). Data are presented as means ± standard error of mean (SEM). Significance was determined using Student’s t-test and denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Data normality was verified prior to the t-test.

Results

Identification of PEBP Gene family members in tobacco and analysis of their protein physicochemical properties

Through HMMER 3.0 analysis and screening via the InterPro online database, 32 PEBP family members were identified in tobacco and named with reference to AtPEBPs. The basic information of the NtPEBP gene family and the physicochemical properties of their encoded proteins are listed in Table 1. The proteins encoded by these genes ranged from 64 to 224 amino acids (aa) in length. The physicochemical properties of the proteins were predicted using the ExPASy online tool. NtTSF5 has the smallest molecular weight of 7469.47, while NtPYBHB4 has the largest molecular weight of 24KDa. The theoretical isoelectric point (pI) ranged from 4.7 (NtFT6) to 10.74 (NtFT10), and the instability index ranged from 22.75 (NtFT8) to 52.09 (NtFT10). The maximum aliphatic index value is 102.72 (NtFT4), and the minimum is 70 (NtPYBHB1). The results of hydrophilicity and hydrophobicity analyses indicate that, except for NtATC1 and NtPYBHB4 proteins which are hydrophobic, the remaining proteins are hydrophilic. Subcellular localization predictions via the Plant-mPLoc online tool show that most NtPEBPs are localized to the nucleus, while NtFT7, NtMFT1, NtPYBHB4, and NtTFL1 are also localized to the cytoplasm, and NtATC1 is additionally localized to the chloroplast. Furthermore, NtATC2, NtATC4, NtPYBHB3, NtBFT1, NtBFT2, NtBFT3, and NtBFT4 are exclusively localized to the cytoplasm (Table 1). Among the 32 NtPEBP genes, 14 are located on 7 chromosomes, while the remaining genes are distributed across multiple genomic scaffolds.

Table 1.

The PEBP genes family members in tobacco

ID	Name	Protein Length(aa)	MW(Da)	Theoretical pI	Homology	Subcellular Location	Chr/scaffold
Nitab4.5_0003472g0020.1	NtATC1	122	13502.53	6.29	AtATC	Chloroplast. Nucleus.	Nitab4.5_0003472
Nitab4.5_0000234g0200.1	NtATC2	173	19637.58	8.54	AtATC	Cytoplasm.	Nt09
Nitab4.5_0008136g0010.1	NtATC3	159	18126.77	8.95	AtATC	Nucleus.	Nitab4.5_0008136
Nitab4.5_0002002g0110.1	NtATC4	171	19473.24	8.97	AtATC	Cytoplasm.	Nitab4.5_0002002
Nitab4.5_0008339g0030.1	NtFT1	146	16566.82	5.31	AtFT	Nucleus.	Nitab4.5_0008339
Nitab4.5_0011525g0020.1	NtFT2	160	17945.32	6.28	AtFT	Nucleus.	Nitab4.5_0011525
Nitab4.5_0008339g0010.1	NtFT3	148	16733.11	5.26	AtFT	Nucleus.	Nitab4.5_0008339
Nitab4.5_0010534g0010.1	NtFT4	125	14532.98	9.71	AtFT	Nucleus.	Nitab4.5_0010534
Nitab4.5_0000971g0160.1	NtFT5	100	11335.08	7.91	AtFT	Nucleus.	Nitab4.5_0000971
Nitab4.5_0000161g0090.1	NtFT6	88	9943.24	4.7	AtFT	Nucleus.	Nt13
Nitab4.5_0000351g0250.1	NtFT7	162	18445.82	7.88	AtFT	Cytoplasm. Nucleus.	Nt22
Nitab4.5_0003848g0050.1	NtFT8	72	7967.04	4.88	AtFT	Nucleus.	Nitab4.5_0003848
Nitab4.5_0006714g0010.1	NtFT9	74	8365.48	5.38	AtFT	Nucleus.	Nitab4.5_0006714
Nitab4.5_0000161g0050.1	NtFT10	96	11077.63	10.74	AtFT	Nucleus.	Nt13
Nitab4.5_0001003g0220.1	NtTSF4	84	9771.1	6.71	AtTSF	Nucleus.	Nt13
Nitab4.5_0018102g0010.1	NtTSF3	86	9780.01	6.82	AtTSF	Nucleus.	Nitab4.5_0018102
Nitab4.5_0008012g0020.1	NtTSF6	155	17674.19	8.71	AtTSF	Nucleus.	Nitab4.5_0008012
Nitab4.5_0001003g0140.1	NtTSF7	175	19791.39	7.86	AtTSF	Nucleus.	Nt13
Nitab4.5_0008012g0010.1	NtTSF5	64	7469.47	6.71	AtTSF	Nucleus.	Nitab4.5_0008012
Nitab4.5_0000751g0180.1	NtTSF1	206	23755.34	8.64	AtTSF	Nucleus.	Nt06
Nitab4.5_0000819g0010.1	NtTSF2	189	21658.92	8.3	AtTSF	Nucleus.	Nt04
Nitab4.5_0000649g0080.1	NtMFT1	184	20451.18	6.6	AtMFT	Cytoplasm. Nucleus.	Nt17
Nitab4.5_0008924g0020.1	NtMFT2	146	16109.19	6.97	AtMFT	Nucleus.	Nitab4.5_0008924
Nitab4.5_0000539g0120.1	NtPYBHB1	142	15681.63	5.93	AtPYBHB	Nucleus.	Nt02
Nitab4.5_0005348g0010.1	NtPYBHB2	168	18563.97	5.23	AtPYBHB	Nucleus.	Nitab4.5_0005348
Nitab4.5_0001986g0140.1	NtPYBHB3	212	23508.41	9.34	AtPYBHB	Cytoplasm.	Nt04
Nitab4.5_0001644g0120.1	NtPYBHB4	224	24752.9	9.22	AtPYBHB	Cytoplasm. Nucleus.	Nt06
Nitab4.5_0003870g0010.1	NtBFT4	174	19325.93	9.13	AtBFT	Cytoplasm.	Nt04
Nitab4.5_0006040g0010.1	NtBFT3	174	19267.89	9.1	AtBFT	Cytoplasm.	Nitab4.5_0006040
Nitab4.5_0001600g0060.1	NtBFT2	174	19357.96	6.98	AtBFT	Cytoplasm.	Nt09
Nitab4.5_0004480g0050.1	NtBFT1	174	19321.01	9.22	AtBFT	Cytoplasm.	Nitab4.5_0004480
Nitab4.5_0005355g0050.1	NtTFL1	158	17569.05	8.68	AtTFL1	Cytoplasm. Nucleus.	Nitab4.5_0005355

Note: This study establishes a new nomenclature for the expanded NtPEBP family. For correspondence with previously published genes, please note: NtFT7 is the primary homologue of the floral repressors NtFT1, NtFT2, and NtFT3 [44]; NtFT2 corresponds to the floral promoter NtFT4 [44]; and NtTSF2 corresponds to the floral promoter NtFT5 [46]

Phylogenetic analysis

To evaluate the phylogenetic relationships among members of the PEBP family, we constructed a phylogenetic tree using PEBP protein sequences from N. tabacum, N. benthamiana, N. sylvestris and N. tomentosiformis, along with previously predicted PEBP protein sequences from Arabidopsis thaliana, pepper, and tomato [35]. The phylogenetic tree is divided into four subfamilies: FT-like, TFL1-like, MFT-like, and YbhB-like (Fig. 1A). In tobacco, the FT-like subfamily consists of 17 members: NtFT1-10, NtTSF1-7. The TFL1-like subfamily comprises 9 members: NtATC1-4, NtBFT1-4, and NtTFL1. The MFT-like subfamily includes 2 members: NtMFT1 and NtMFT2. Additionally, 4 members belong to the YbhB-like subfamily: NtPYBHB1-4 (Fig. 1A). The NtPEBP protein sequences were subjected to multiple sequence alignment using ClustalW, and the resulting alignment was trimmed using the trimAl tool [67]. Results indicate that most NtPEBP proteins possess interaction sites with 14-3-3 proteins (RXF motif). Furthermore, the majority of these proteins also contain DPDxP and GxHR motifs (Fig. 1B).

Fig. 1.

Phylogenetic Analysis and Multiple Sequence Alignment of NtPEBP Proteins. (A) Phylogenetic tree: The tree includes 32 NtPEBP genes, 24 NbPEBP genes, 16 NsPEBP genes, 20 NtoPEBP genes, 7 AtPEBP genes, 12 SlPEBP genes, and 11 CaPEBP genes. Different colors represent different PEBP subfamilies. Blue represents the TFL1-like subfamily, red represents the MFT-like subfamily, green represents the FT-like subfamily, and yellow represents the YbhB-like subfamily. Different symbols represent different species. Square represents Nicotiana tabacum, circle represents Nicotiana benthamiana, outer triangle represents Nicotiana sylvestris, inner triangle represents Nicotiana tomentosiformis, star represents Arabidopsis thaliana, check represents Solanum lycopersicum and hollow circle represents Capsicum annuum. (B) Multiple sequence alignment of 32 NtPEBP protein sequences. The red-marked regions at the bottom indicate the anion binding sites

To further evaluate the evolutionary constraints acting on the NtPEBP gene family, we calculated the non‑synonymous (Ka) and synonymous (Ks) substitution rates for representative paralogous gene pairs identified within the family (Table S2). The Ka/Ks ratios of all analyzed duplicated pairs were below 0.6, ranging from 0.095 (NtBFT2/NtATC4) to 0.578 (NtTSF3/NtTSF4), with an average of 0.274. Among these, 41.9% (13/31) of the pairs exhibited Ka/Ks ratios between 0.1 and 0.2, 12.9% (4/31) between 0.2 and 0.3, 12.9% between 0.3 and 0.4, 12.9% between 0.4 and 0.5, and 12.9% between 0.5 and 0.6; only 6.5% (2/31) showed ratios below 0.1. Notably, two gene pairs (NtFT3/NtFT2 and NtFT5/NtFT4) had Ks values of zero, indicating extremely recent duplication events. Collectively, these results suggest that the NtPEBP gene family has mainly evolved under strong purifying selection, with functional conservation prevailing after duplication.

Prediction of the three-dimensional structure of the NtPEBP protein

The three-dimensional structure of a protein is intrinsically linked to its biological function. Using the AlphaFold Server, we predicted the structures of all 32 NtPEBP proteins. All members share a conserved fold consisting of antiparallel β-sheets surrounded by α-helices, though distinct variations were observed among different classes. Within the NtATC class, the number of β-sheets ranges from 5 in NtATC3 to 8 in NtATC4, with the majority containing 7. With respect to α-helices, NtATC1 possesses three, while all other NtATC proteins contain five. All NtBFT proteins adopt a conserved architecture featuring eight β-sheets and five α-helices. Substantial structural diversity was observed among NtFT members: NtFT10 contains only 2 β-sheets; NtFT8 and NtFT9 have 3; NtFT5 has 4; NtFT1 and NtFT4 possess 6; and the remaining members contain 5 β-sheets. All NtFT proteins contain between 2 and 4 α-helices. In the NtMFT class, NtMFT1 consists of 7 β-sheets and 4 α-helices, whereas NtMFT2 has 4 β-sheets and 3 α-helices. The NtPYBHB class exhibits considerable structural heterogeneity: NtPYBHB1 to NtPYBHB4 contain 5, 6, 7, and 10 β-sheets, respectively; NtPYBHB1 and NtPYBHB4 contain 4 α-helices, while NtPYBHB2 and NtPYBHB3 contain 5. Among NtTSF proteins, NtTSF3 to NtTSF5 contain only 2 β-sheets, NtTSF6 contains 5, and the remaining members contain 7. The α-helical count also varies, with NtTSF3 and NtTSF4 containing 2 helices, NtTSF5 containing 1, and the rest containing 3 to 4 (Fig. 2).

Fig. 2.

Three-dimensional structure of NtPEBP family member proteins. Blue represents α-helices, purple represents β-sheets, and brown represents random coils

Structural and conserved motif analysis of NtPEBPs

A phylogenetic tree comprising only the NtPEBP family genes was constructed using IQ-TREE 2. This tree divided the subfamily into four distinct clades, a result consistent with the previously constructed evolutionary tree. Based on this phylogenetic tree, we further analyzed the conserved motifs and gene structures of the NtPEBP gene family members (Fig. 3).

Fig. 3.

Gene structure and conserved motifs of the NtPEBP family members in tobacco. (A) Phylogenetic tree of the NtPEBP gene family members. (B) Distribution and order of conserved motifs in NtPEBP proteins. Different motifs are indicated by distinct colored boxes. (C) Gene structures of the NtPEBP family members in tobacco. Green boxes represent untranslated regions (UTRs), while yellow boxes represent coding sequences (CDSs). The scale bar at the bottom is provided for coFmparing the lengths of different genes and proteins. (D) Four major motif logo of NtPEBPs and the character sequence of these motif

In the analysis of conserved motifs, 10 motifs were identified and designated as Motif 1 to Motif 10. NtTSF5 contains 1 motif; NtFT10 and NtTSF4 contain 2 motifs; NtATC1, NtFT4, NtFT5, NtFT6, NtFT8, NtTSF3, NtPYBHB1, and NtPYBHB2 contain 3 motifs; NtTSF6 and NtMFT1 contain 4 motifs; NtATC3, NtATC4, NtFT1, NtFT3, and NtBFT4 contain 5 motifs; while the remaining family members contain 6 motifs. Gene structure analysis revealed that 11 genes contain 2 exons, 4 genes contain 3 exons, 1 gene contains 5 exons, and the remaining genes contain 4 exons.

Analysis of cis-acting elements in NtPEBPs

The cis-acting elements in promoters often regulate gene expression and function. Various cis-acting elements were identified in the promoters of PEBP genes, which can be categorized into four main types: hormone-responsive elements, light-responsive elements, stress-responsive elements, and development-related elements (Fig. 4).

Fig. 4.

Prediction and analysis of cis-acting elements in the promoter regions (-1500 bp) of the NtPEBP gene family. (A) Distribution of cis-acting elements in the promoter regions (-1500 bp) of NtPEBP genes. Different colors represent distinct categories of cis-acting elements: “Growth” indicates development-related elements, “Hormones” represents hormone-responsive elements, “Light” denotes light-responsive elements, and “Stress” refers to stress-responsive elements. The scale bar at the bottom indicates the sequence direction and length. (B) Classification and statistics of cis-acting elements. The elements are categorized into four major types. The numbers in the grid indicate the count of elements within each major category, while the blue-to-red color gradient reflects the quantitative distribution from low to high

Regarding hormone-related cis-acting elements, we identified hormone-responsive elements such as TGACG-motif, TCA-element, CGTCA-motif, GARE-motif, and ABRE in the promoter regions of tobacco PEBP genes. Light-responsive elements included Box 4, I-box, G-box, GT1-motif, and GATA-motif. Stress-responsive elements comprised MBS, O2-site, TC-rich repeats, ARE, MYB, and MYC. Additionally, we identified development-related elements, such as CAT-box, GCN4_motif, HD-Zip 1, RY-element, and AACA_motif. Detailed categories of cis-acting elements in the promoter regions of NtPEBP genes are listed in Table S3. Among the 32 NtPEBP genes, except for NtATC2, NtFT2, NtFT6, NtFT3, NtTSF1, NtPYBHB1, NtBFT2, and NtBFT1, whose promoter regions lack development-related elements, all other family members contain hormone-responsive, light-responsive, stress-responsive, and development-related elements (Fig. 4). Detailed information on the cis-acting elements in the promoter regions of NtPEBP genes is provided in Table S4.

Tissue-specific expression profile of NtPEBPs

Analysis of publicly available RNA-seq data revealed the expression profiles of all 32 NtPEBP genes across various tissues and developmental stages (Fig. 5). Members of the MFT-like and YbhB-like subfamilies were predominantly expressed in dry capsules; notably, NtPYBHB1 and NtPYBHB2 also showed marked expression in young flowers. Within the TFL1-like subfamily, all members except NtATC1 were highly expressed in roots. Among the FT-like members, NtTSF4 exhibited high transcript levels in young leaves, whereas NtTSF1 and NtTSF2 were predominantly expressed in stems, and NtFT6 and NtFT7 were specifically detected in leaves. The remaining NtPEBP genes showed low or undetectable expression across the examined tissues. Detailed TPM values are provided in Table S5.

Fig. 5.

Expression patterns of NtPEBP genes in different tissues and developmental stages of tobacco. Heatmap was generated by row-normalizing the TPM (Transcripts Per Million) values of each gene across homologous tissues and developmental stages. Expression levels are represented by the color scale (log2-transformed)

Expression of NtPEBPs in different tissues

To further investigate the tissue-specific expression patterns of NtPEBPs, qRT-PCR was performed to analyze the transcript levels of all NtPEBP members in roots, stems, leaves, and flowers tissues. Genes with undetectable or minimal expression, including NtFT1, NtFT9, NtFT10, NtPYBHB3, NtPYBHB4, NtTSF5, NtTSF6, and NtTSF7 were excluded from the figure. Analysis of the expressed genes revealed distinct spatial patterns (Fig. 6). Specifically, NtATC1/2 were highly expressed in roots, whereas NtATC3/4 showed relatively lower expression in roots but were predominantly expressed in stems and flowers (Fig. 6A, B). Several NtBFT homologs were detected at higher levels in stems (Fig. 6C). The expression patterns of NtFT2 to NtFT8 were similar, with primary expression in leaves and low or undetectable levels in other tissues (Fig. 6D–H). NtMFT1/2 were expressed across multiple tissues with significant differences, showing the highest transcript accumulation in stems (Fig. 6I). NtPYBHB1/2 expression was significantly higher in leaves and flowers than in roots (Fig. 6J). Both NtTFL1 and NtTSF1/2 were highly expressed in stems and flowers (Fig. 6K, L), while NtTSF3 and NtTSF4 expression was predominantly detected in leaves (Fig. 6M, N). Although the sampling stage differed from that of the transcriptome data, the expression patterns of multiple genes were largely consistent with the RNA-seq results (Figs. 5 and 6).

Fig. 6.

Relative expression level of NtPEBPs in tobacco. (A) NtATC1/2^$, (B) NtATC3/4^$, (C) NtBFT1/2/3/4^$, (D) NtFT2/3^$, (E) NtFT4/5^$, (F) NtFT6, (G) NtFT7, (H) NtFT8, (I) NtMFT1/2^$, (J) NtPYBHB1/2^$, (K) NtTFL1, (L) NtTSF1/2^$, (M) NtTSF3, (N) NtTSF4. ND, Not Detected. All data are presented as the mean (± SEM) of three independent biological determinations and were analyzed by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). ^$, Primers do not distinguish between tobacco homeologs

Subcellular localization

Subcellular localization predictions via Plant-mPLoc indicated that NtATC4, NtFT7, NtBFT1, and NtBFT4 are all localized to the cytoplasm. Additionally, NtFT7 is also localized to the nucleus. Transient expression of NtATC4-GFP, NtFT7-GFP, NtBFT1-GFP, and NtBFT4-GFP fusion proteins was performed in leaves of N. benthamiana. Fluorescence analysis results demonstrated that the subcellular localization patterns of the NtATC4-GFP, NtFT7-GFP, NtBFT1-GFP, and NtBFT4-GFP fusion proteins were consistent with the earlier predictions (Fig. 7).

Fig. 7.

Subcellular localization of NtATC4, NtFT7, NtBFT1, and NtBFT4 in epidermal cells of N. benthamiana leaves. GFP: Green fluorescent protein; DAPI: Fluorescent dye that strongly binds to DNA; Bright: Bright-field image; Merged: Overlay of GFP, DAPI and Bright signals. Scale bar: 50 μm

Interaction between NtPEBPs protein and Nt14-3-3 Most members of the PEBP family can interact with 14-3-3 proteins to form complexes. By binding to 14-3-3 proteins and the bZIP transcription factor FD, PEBP family members constitute the florigen activation complex (FAC), which subsequently activates meristem-specific genes such as AP1 [68]. The interactions between four members of the NtPEBP gene family (NtATC4, NtFT7, NtBFT1, and NtBFT4) and the Nt14-3-3 protein were analyzed using a yeast two-hybrid assay. The results demonstrated that NtATC4, NtFT7, NtBFT1, and NtBFT4 can interact with the Nt14-3-3 protein (Fig. 8A). Furthermore, bimolecular fluorescence complementation assay in tobacco cells showed green fluorescence in each case when NtATC4, NtFT7, NtBFT1, or NtBFT4 was co-expressed with the Nt14-3-3 protein (Fig. 8B).

Fig. 8.

Interactions between NtATC4, NtFT7, NtBFT1, and NtBFT4 with Nt14-3-3. (A) Yeast two-hybrid assay. BD-53 + AD-T and BD + AD were used as positive and negative controls, respectively. Yeast co-transformants were first cultured on SD/-Leu/-Trp medium [double-dropout medium (DDO), lacking leucine and tryptophan], and subsequently transferred to SD/-Leu/-Trp/-His/-Ade medium [quadruple-dropout medium (QDO), lacking leucine, tryptophan, histidine, and adenine] for further cultivation. (B) bimolecular fluorescence complementation assay. YFP: Green fluorescent protein; Bright: Bright-field image; Merged: Overlay of YFP and Bright signals. Scale bar: 50 μm

Silencing of NtATC4 affects tobacco development and delays flowering.

To determine the functional role of NtATC4 in tobacco, we employed virus-induced gene silencing (VIGS) to knock down its expression. Compared with control plants, NtATC4-silenced tobacco lines exhibited enhanced elongation of the main stem and increased leaf surface area (Fig. 9A, B, F, G). Furthermore, at 25 days post-silencing, visible flower buds had emerged in both wild-type (WT) and negative control plants, whereas no flowering was observed in the NtATC4-silenced lines (Fig. 9C). Reverse transcription PCR (RT-PCR) analysis confirmed a significant downregulation of NtATC4 expression in the silenced plants compared with the negative controls, validating the effectiveness of the gene silencing procedure (Fig. 9D, E). These results collectively indicate that NtATC4 may plays a regulatory role in both vegetative growth and floral transition in tobacco (Fig. 9F-H). Furthermore, we analyzed the expression of flowering-related genes, including AP1, CO, LFY, SOC1, and SVP homology genes in N. benthamiana. In NtATC4-silenced plants, the transcript level of NbAP1 was elevated (Fig. 9I), whereas NbCO and NbLFY expression showed no significant change (Fig. 9J, K). By contrast, NbSOC1 expression was significantly downregulated (Fig. 9L), and NbSVP expression was markedly upregulated (Fig. 9M).

Fig. 9.

Silencing of NtATC4 in Nicotiana benthamiana using a tobacco rattle virus (TRV)-based VIGS system. (A) The phenotype of tobacco (N. benthamiana) at the seedling stage after silencing of NtATC4 using VIGS technology. Scale bars, 5 cm. (B) The single seedling phenotype after silencing using VIGS technology. Scale bars, 5 cm. TRV::PDS are positive control, TRV:00 are blank control. (C) Flowering status 25 days after gene silencing. (D) Expression level of NtATC4 after VIGS detected by RT-PCR. Nt26s was used as a reference gene. (E) Expression level of NtATC4 after VIGS detected by qRT-PCR. Nt26s was used as a reference gene. (F) Days to flower bud emergence from sowing. (G) Statistics on the length of the main stems of plants after different treatments. The main stem length was measured from the first node at the base to the axil of the uppermost fully expanded leaf. (H) Statistics on the surface area of plant leaves after different treatments. The third fully expanded leaf from the apical meristem downward was measured for each treatment. (I-M) The expression levels of NbAP1 (I), NbCO (J), NbLFY (K), NbSOC1 (L), and NbSVP (M) in NtATC4-silenced plants by qRT-PCR. The species prefix “Nb” is omitted from gene labels for simplicity. All data are expressed as the mean ± standard error of the mean and analyzed using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). ns, not significant

Discussion

Tobacco yield is closely related to plant architecture and flowering time. Currently, mechanized and automated harvesting is gradually replacing manual operations, thereby reducing economic and time costs. Research on tobacco plant architecture is of great significance to meet the demands of mechanized harvesting. The plant PEBP gene family plays a central role in developmental processes, such as floral transition, plant morphological development, and seed germination [17, 69, 70]. Studying the tobacco PEBP gene family provides important guidance for breeding tobacco varieties suitable for mechanized harvesting. However, no systematic and comprehensive study on the tobacco PEBP gene family has been reported yet.

In N. tabacum, N. benthamiana, N. sylvestris, and N. tomentosiformis, 32, 24, 16, and 20 PEBP genes were identified, respectively, and classified into four subfamilies: FT-like, TFL1-like, MFT-like, and YbhB-like. Our study includes several previously uncharacterized members, particularly within the FT-like and TFL1-like subfamilies. The FT-like subfamily has the largest number of members in tobacco, suggesting it may play more complex and diverse roles in regulating tobacco plant architecture and flowering. The larger number of PEBP family members in tobacco compared to other Solanaceae species may be due to the fact that tomato, pepper, potato, and petunia are diploids, whereas tobacco is an allotetraploid formed by the hybridization of N. sylvestris and N. tomentosiformis [71–73]. The number of PEBP genes in N. tabacum is lower than the sum of those in its progenitor species, N. sylvestris and N. tomentosiformis, which may be due to gene loss or pseudogenization following allopolyploidization. Owing to the incomplete chromosome-level assembly of the tobacco genome, only tandem duplications were identified within the NtPEBP gene family. Specifically, a single tandem duplicate pair, NtFT1 and NtFT3, was found. This pair exhibited a Ka/Ks ratio of 0.51, indicating strong purifying selection and limited functional divergence. The low Ks value (0.115) further suggests that this tandem duplication event occurred relatively recently (Table 2).

Previous studies have demonstrated that exon-intron structures are involved in the evolution of plant species [74]. Gene structure analysis revealed that NtPEBP genes contain 2 to 5 exons, with half of them possessing 4 exons (Fig. 3), which is consistent with observations in PEBP gene families of other species [35, 75, 76]. Half of the NtPEBP gene family members exhibit a gene structure composed of four exons and three introns, with similar conserved motif distribution patterns (Fig. 3), indicating evolutionary conservation among these genes. Distinct differences in exon numbers and motif composition were observed among members of different subfamilies, likely resulting from evolutionary divergence. The short motifs DPDxP (Asp-Pro-Asp-X-Pro) and GxHR (Gly-x-His-Arg) are highly conserved and represent the characteristic motifs of the PEBP protein family [77]. The conserved protein motif identifies motif1 and motif4 as the characteristic motifs of N. tabacum. A subset of PEBP genes lack Motif 4 (Fig. 3). These structural variations may be associated with their functional differentiation. Mutations or deletions in the GxHR motif may affect their binding to phosphate ions, thereby altering their interaction with FD (FLOWERING LOCUS D) [78].

The core promoter and its associated cis-acting elements are crucial for transcriptional regulation of genes, as they serve as binding sites for specific proteins [79–81]. Analysis of cis-acting elements in the promoters of tobacco PEBP gene family members revealed that each gene contains multiple types of promoter cis-acting elements, which were categorized into four major classes: hormone-responsive elements, light-responsive elements, stress-responsive elements, and development-related elements. Except for NtATC2, NtFT3, NtFT6, NtTSF1, NtPYBHB1, NtBFT1, and NtBFT2, which lack development-related elements, all other NtPEBP family members possess all four types of cis-acting elements (Fig. 4). Light-responsive elements are the most abundant in the NtPEBP gene family, indicating that the functions of these genes are primarily associated with light response and that their expression is regulated by light. Furthermore, the NtPEBP gene family is involved in hormone response, flowering time regulation, abiotic stress response, and plant growth and development. RNA-seq data analysis revealed that some NtPEBP family members exhibit tissue-specific expression (Fig. 5). NtMFT1, NtMFT2, NtPYBHB3, and NtPYBHB4 are specifically expressed in dry capsules, suggesting their potential roles in seed development and germination. Additionally, NtPYBHB1 and NtPYBHB2 are expressed not only in dry capsules but also in young flowers, indicating that these two genes may also regulate flowering or development of flower in tobacco. Several genes in the TFL-like subfamily of tobacco are primarily expressed in roots, possibly associated with root development and growth regulation. PEBP genes play important roles in various tissues of tobacco.

Prior functional studies have identified specific PEBP members as key floral regulators: three as inhibitors and two as promoters [44, 46]. Our analysis reveals that the three previously reported repressors are all homologous to the single gene we designated NtFT7 [44]. This suggests that NtFT7 is the key ortholog underlying the floral inhibition previously attributed to multiple genes. Conversely, the previously reported promoters correspond to our NtFT2 and NtTSF2 [44, 46]. The expression patterns we observed corroborate their proposed functions: both NtFT2 and NtTSF2 were highly expressed in leaves (Fig. 6), the primary site of florigen production, which is consistent with their roles as flowering promoters.

The 14-3-3 proteins constitute a family of small regulatory molecules involved in various plant processes, including flowering, growth, metabolism, and immune responses [82]. PEBP proteins influence plant flowering by forming complexes with 14-3-3s [32]. In this study, yeast two-hybrid assays were employed to verify the interaction between a specific tobacco 14-3-3 member, Nt14-3-3, and four NtPEBP proteins. However, it remains unknown whether other members of the tobacco 14-3-3 family also interact with NtPEBP proteins and regulate flowering in tobacco. Most interactions with 14-3-3 proteins contain the following motifs: RSXpSXP [83], RXSXpSXP [84], RXF/YpSXP [85], and YpTV [86]. Multiple sequence alignment revealed that the RXF motif is conserved across most NtPEBP proteins. This conservation suggests that these NtPEBP proteins may also interact with other members of the Nt14-3-3 family. Our Y2H and BiFC assays confirmed that NtATC4, NtFT7, NtBFT1, and NtBFT4 all interact with Nt14-3-3 proteins, and that these interactions occur in the cell wall. This suggests their potential involvement in a module analogous to the Florigen Activation Complex (FAC). However, the functional outcomes of these interactions appear to depend on the specific PEBP family member. In Arabidopsis and rice, FT-like proteins typically promote flowering by forming an active FAC with 14-3-3 and FD, whereas certain TFL1-like proteins act as competitive antagonists to repress flowering [26, 87]. Intriguingly, despite its phylogenetic classification as a TFL1-like protein, our functional data indicate that NtATC4 acts as a promoter of flowering in tobacco. This conclusion is supported by our VIGS results, in which silencing NtATC4 significantly delayed flowering and enhanced vegetative growth—a phenotype opposite to that of its Arabidopsis homolog ATC, which functions as a floral repressor when overexpressed [25]. In Arabidopsis, TFL1 delays flowering by repressing the expression of AP1 and LFY, while SVP functions similarly by suppressing SOC1 expression. AP1, LFY, and SOC1 are floral promoters, whereas SVP is a floral repressor [88]. In NtATC4-silenced plants, downregulation of NtATC4 leads to a slight upregulation of AP1 and LFY expression. However, in a parallel flowering pathway, the expression of SVP is significantly increased, accompanied by a marked decrease in SOC1 expression. Collectively, these opposing expression changes may tip the balance toward floral repression and lead to the delayed flowering phenotype.

This functional divergence highlights a potential species-specific evolution of ATC genes within the Solanaceae. The loss of the flowering promoter NtATC4 likely disrupts the precise balance of flowering signals, delaying the floral transition and potentially reallocating resources toward vegetative growth. Although our VIGS data support a flowering-promoting role for NtATC4 in tobacco, we cannot rule out indirect effects. Heterologous complementation and spatial expression analysis are required to validate this functional divergence conclusively.

Conclusions

In this study, 32 members of the PEBP gene family were identified and screened from the tobacco genome, and these genes were classified into four subfamilies: FT-like, TFL1-like, MFT-like, and YbhB-like. We further analyzed the gene structures, conserved motifs, physicochemical properties, and expression patterns of NtPEBPs. Subsequently, qRT-PCR was employed to investigate the expression profiles of NtPEBPs in depth, revealing their potential roles in tobacco plant architecture, growth, development, and flowering. Among them, interactions between NtATC4, NtFT7, NtBFT1, and NtBFT4 with Nt14-3-3 were confirmed, suggesting that these genes may form complexes with Nt14-3-3 proteins to modulate flowering in tobacco. In summary, this work provides an annotated resource for the NtPEBP gene family, including phylogeny, motif architecture, promoter elements, and expression landscapes. The validated NtPEBP–14-3-3 interaction modules, exemplified by the floral activator NtATC4, offer entry points for engineering flowering time and vegetative growth in tobacco.

Authors’ contributions

S.Q. and G.D.P. designed the study. S.Q. and L.X.H. carried out bioinformation analyses. S.M.M. and Y.M.F. provided the tobacco materials. Z.Z.B., L.X.H., X.W.X., C.X.X. and Z.L.L. collected plant materials and carried expriments. XMM., C.M.L., and L.Z.Y. carried out other experiments. L.X.H. and S.Q. wrote the original draft. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by The Key Science and Technology Project of China National Tobacco Corporation [grant No.110202201007(JY-07)].

Data availability

The following information was supplied regarding data availability: Data are available at NCBI SRA: SRX108496, SRX108498–SRX108502.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.