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Article

Genome-Wide Identification and Characterization of BPC Transcription Factors in Tobacco (Nicotiana tabacum)

by
Ying Zhang
1,*,†,
Shengwu Jia
2,†,
Sisi Wang
1,
Xiaolian Ji
1 and
Zijin Liu
2,*
1
College of Ecological and Environmental Engineering, Shaanxi A&F Technology University, Yangling 712100, China
2
Shaanxi Key Laboratory of Crop Heterosis, College of Agronomy, Northwest A&F University, Yangling 712100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(9), 2084; https://doi.org/10.3390/agronomy15092084
Submission received: 30 July 2025 / Revised: 26 August 2025 / Accepted: 27 August 2025 / Published: 29 August 2025
(This article belongs to the Section Crop Breeding and Genetics)
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Abstract

BASIC PENTACYSTEINE (BPC) is a small family of plant-specific transcription factors that play crucial roles in plant growth, development processes, and response to abiotic stresses. However, the specific roles of Nicotiana tabacum BPCs (NtBPCs) remain ambiguous. Here, we identified 12 NtBPC genes, 5 of which were mapped to four chromosomes. Phylogenetic analysis classified these genes into three subfamilies. Collinearity was observed among BPC genes of N. tabacum, Capsicum annuum, and Solanum lycopersicum. Moreover, polypeptides encoded by NtBPC genes within the same subfamily shared similar conserved motifs and protein domains. Subcellular localization showed that 10 NtBPC proteins are localized in the nucleus. Promoter analysis revealed the presence of abiotic stress response elements in the promoters of NtBPCs. Further tissue-specific expression analysis using RT-qPCR revealed that NtBPCs are highly expressed in stems and leaves. After drought, NaCl, and cold treatments, NtBPCs exhibited varied expression patterns. These findings provide valuable insights into the evolutionary dynamics of the NtBPC gene family and lay the groundwork for subsequent investigations into the functions of NtBPC genes.

1. Introduction

Tobacco (Nicotiana tabacum L.) is an important leaf economic crop. Throughout its growth cycle, tobacco often faces various adverse stresses, such as pathogenic bacteria, drought, low temperature, salinization, and heavy metals [1,2]. In recent years, the growing demand for tobacco leaves has highlighted the necessity of genetic improvements to enhance leaf quality, expand leaf area, and strengthen tolerance to both biotic and abiotic stresses. Environmental stressors like drought, salt (NaCl), and low temperature have been increasingly detrimental to plant growth and development, leading to reduced yield and quality in crops [3]. Hence, there is a pressing need for functional studies on tobacco stress responses and identification of stress-related genes to unravel the molecular mechanisms underpinning tobacco stress tolerance and shield the crop from detrimental environmental impacts.
BASIC PENTACYSTEINE/BARLEY B RECOMBINANT (BPC/BBR) is a plant-specific transcription factor (TF) family that plays a crucial role in gene transcription regulation [4]. In Arabidopsis thaliana, seven BPC genes have been identified and categorized into three classes: Class I (AtBPC1~AtBPC3), Class II (AtBPC4~AtBPC6), and Class III (AtBPC7). With the exception of AtBPC5 that is defined as a pseudogene, the other six AtBPC genes encode either activators or repressors of transcription [4,5]. In vitro studies have demonstrated that AtBPC1 binding induces DNA bending in the AtSEEDSTICK promoter region, a process potentially facilitated by AtBPC1’s ability to oligomerize and bind multiple GAGA fragments within this promoter [6,7,8]. AtBPC2 has been shown to negatively regulate the expression of AtLATE EMBRYOGENSIS ABUNDANT4-5, thereby influencing osmotically induced H2O2 accumulation, malondialdehyde content, and electrolyte leakage percentage [9]. Ectopic expression of AtBPC3 hinders leaf margin formation by inhibiting the expression of AtTEOSINTE BRANCHED1, AtCYCLOIDEA and AtPROLIFERATING CELL FACTORs [10]. AtBPC1, AtBPC2, AtBPC4, and AtBPC6 can regulate lateral root development by directly inhibiting AtABSCISIC ACID INSENSITIVE4 (AtABI4) expression [11]. Emerging studies also indicate that BPCs are involved in regulating plant responses to hormones, like ethylene and cytokinin [5,12,13,14]. The completion of genome sequencing in various species has enabled the identification and characterization of numerous genes. The BPC TF family has been previously identified in plant species like Oryza sativa, Cucumis sativus, Hordeum vulgare, Glycine max and Brassica napus [15,16,17,18,19]. However, there is a lack of comprehensive analysis of the NtBPC family, and the functions of NtBPC members are still unknown.
The objectives of this study are to provide essential information and insights into the evolutionary dynamics and potential functions of the NtBPC gene family. Here, 12 NtBPCs were identified from the tobacco genome using bioinformatics approaches, followed by comprehensive analyses of the NtBPC gene family, including phylogenetic relationships, gene structures, conserved motifs, and gene expression patterns. Overall, these findings enhance our understanding of NtBPCs and lay a foundation for further functional analysis, gene editing, and genetic engineering of NtBPC genes.

2. Materials and Methods

2.1. Plant Growth Conditions and Stress Treatments

The experiment utilized cultivated tobacco ‘K326’ (N. tabacum, 4.5 Gb genome) grown in controlled conditions within an incubator set at a temperature of 26 °C, humidity ranging from 70% to 75%, with a light cycle of 16 h on and 8 h off. The daytime light intensity was maintained at 300 μmol·m−2·s−1. Different tissues of the tobacco including roots, stems, leaves, and shoot apical meristems were harvested in vigorous growth stage (90 days after sowing), quickly frozen in liquid nitrogen, and stored at −80 °C for further analysis.
Tobacco seeds were sterilized and sown on Murashige and Skoog (MS) solid medium. Once the seedlings reached two true leaves in an artificial climate chamber, those exhibiting uniform growth were transplanted into pots and cultivated in a greenhouse. Upon reaching four true leaves, the seedlings underwent the treatments of drought, 200 mM NaCl, and cold (4 °C). For cold treatment, seedlings were transplanted into artificial climate chamber maintained at 4 °C. Seedlings at the same stage were watered with 16% PEG6000 and 200 mM NaCl solution, followed by sampling at 2, 3, 4, and 5 days (D), which simulated the drought and salt stress, respectively. Untreated plantlets served as controls. Three independent replications were conducted for each stress condition, with three tobacco seedlings of consistent growth selected for each replication. Total RNA was extracted by quick-freezing the leaves in liquid nitrogen.

2.2. Identification and Physicochemical Property Anaylsis of NtBPCs

The protein sequences of AtBPC family members were retrieved from the TAIR database (https://www.arabidopsis.org/, accessed on 10 June 2025). Homology searches against the tobacco (Nicotiana tabacum) genome (assembly available at https://solgenomics.net/organism/Nicotiana_tabacum/genome, accessed on 10 June 2025) were performed using BLASTP with an E-value cutoff of 5. The obtained sequences were analyzed for their conserved domains in the Pfam (http://pfam.xfam.org/, accessed on 11 June 2025) database [20]. Because all BPC family members contain GAGA domains, sequences without GAGA domains were removed. Use the online websites ExPASy (https://www.expasy.org/, accessed on 12 June 2025) and Wolf PSORT (https://wolfpsort.hgc.jp/, accessed on 12 June 2025) to predict amino acid length, protein molecular weight, isoelectric point, instability index and subcellular localization [21].

2.3. Phylogenetic Tree

The neighbor-joining method (Parameters: Poisson correction, pairwise deletion and 1000 repeated bootstrap) [22] was used to conduct evolutionary tree of the BPC proteins in MEGA X (v5.5.8, accessed on 12 June 2025). Use the online software Itol (v7.2.1, https://itol.embl.de/itol.cgi, accessed on 12 June 2025) to beautify the evolutionary tree. NtBPC family members are classified with reference to AtBPC family members.

2.4. Chromosomal Location and Collinearity Analysis of NtBPCs

Retrieve the chromosomal location of the NtBPC genes from the tobacco genome annotation data. The collinearity analysis was conducted by using TBtools (v2.210, accessed on 13 June 2025), and visualizations of the comparison was created by TBtools (v2.210, accessed on 13 June 2025) [23].

2.5. Gene Structure, and Conserved Motif Analyses

The gene structures of NtBPC genes were analyzed using TBtools (v2.210, accessed on 15 June 2025) [23]. Subsequently, we conducted motif analysis using the MEME program (v5.5.8, https://meme-suite.org/meme/tools/meme, accessed on 16 June 2025), with a maximum of 10 motifs and an optimal motif width of 6–200 amino acid residues, while keeping the remaining settings at default values [24]. Next, we employed the Batch CD-Search function on NCBI (https://www.ncbi.nlm.nih.gov, accessed on 16 June 2025) to identify the conserved domain of NtBPCs.

2.6. Cis-Element Analysis of NtBPC Gene Promoters

The 2000 bp promoter sequence located upstream of the start codon of each NtBPC gene was retrieved from the standard tobacco reference genome. Subsequently, this sequence was analyzed using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 18 June 2025) for cis-element prediction. The predicted cis-elements were then visualized using the TBtools (v2.210, accessed on 18 June 2025).

2.7. Transient Expression in N. benthamiana Leaves

Recombinant plasmids containing the GFP reporter gene were constructed by amplifying and cloning the coding domain sequences (CDSs) of NtBPCs, excluding the stop codons, into the pCAMBIA-1300–35S–GFP vector. For subcellular localization assays, the recombinant plasmids with the GFP reporter gene were introduced into A. tumefaciens strain GV3101. The transformed bacteria were injected into the leaves of 3-week-old N. benthamiana seedlings from the abaxial side using a sterile syringe. Transient expression was observed following the established protocol, and images were captured using a laser scanning confocal microscope (LSM 700; ZEISS, Oberkochen, Germany) at 72 h post agroinfiltration [25]. All primers used for gene cloning are listed in (Supplementary Table S1).

2.8. RNA Isolation and RT-qPCR

RNA was extracted from various tissues of tobacco and treated leaves using the MiniBEST Plant RNA Extraction Kit (TaKaRa Bio, Dalian, China) and reverse transcribed with PrimeScript RT Mix (TaKaRa Bio, Dalian, China). RT-qPCR analysis was conducted using the Quant Studio 7 Real Time system and SYBR Green Master Mix (Cofitt, Hong Kong, China). The expression levels of the target genes were normalized to NtHSC70-1 gene expression using the 2−ΔΔCt method [26]. Each gene was analyzed with three independent biological replicates. Gene-specific primers for RT-qPCR are provided in Supplementary Table S1.

3. Results

3.1. Identification and Physicochemical Property Analysis of NtBPCs

A BLASTP search was conducted in the tobacco genome database using the protein sequences of AtBPC members as queries to identify potential NtBPC members. A total of 12 NtBPC members were identified after analyzing the conserved domains of the candidate members through the Pfam database. They were denoted as NtBPC1 to NtBPC12, respectively (Supplementary Table S2). Chromosomal localization analysis showed that only 5 NtBPC genes were identified on the tobacco chromosomes. Specifically, NtBPC6 and NtBPC12 were mapped onto chromosome 23, while NtBPC4, NtBPC5, and NtBPC8 were located on chromosomes 21, 12, and 17, respectively (Figure 1).
The average length of NtBPC proteins is 320 amino acids, with the longest being 404 amino acids (NtBPC12) and the shortest being 282 amino acids (NtBPC1 and NtBPC2) (Supplementary Table S3). Analysis of the physicochemical properties of NtBPC proteins showed that their molecular weights ranged from 31.29 kDa (NtBPC1) to 44.53 kDa (NtBPC12) (Supplementary Table S3). The theoretical isoelectric points (pI) of these proteins range from 7.79 to 9.62, and the pI of all NtBPCs is greater than 7, indicating that NtBPCs are positively charged under physiological conditions. Proteins with an instability index exceeding 40 are classified as unstable proteins [27]. The instability indices of NtBPC proteins ranged from 42.88 to 62.58 (Supplementary Table S3), indicating that all NtBPC proteins are unstable. Meanwhile, subcellular localization prediction showed that except for NtBPC8 and NtBPC11 were separately localized in chloroplast and cytosol, other 10 NtBPC proteins were localized in the nucleus (Supplementary Table S3), which was also confirmed by subcellular localization assays (Figure 2). These results suggest that these 10 proteins may function as TFs.

3.2. Phylogenetic and Genomic Synteny Analysis of NtBPC Proteins

To examine the evolutionary relationship of BPC members, we constructed a phylogenetic tree using the full-length amino acid sequences of BPC proteins from A. thaliana and N. tabacum. The 12 NtBPC proteins were categorized into 3 subfamilies (Figure 3), consistent with previous classifications in A. thaliana [5]. Specifically, 4 NtBPC proteins were assigned to subfamily I, 7 to subfamily II, and 1 to subfamily III (Figure 3).
To explore the evolutionary mechanisms of the BPC family members in Solanaceae species, we conducted the collinearity analysis of BPC genes among Solanum lycopersicum, Capsicum annuum, and N. tabacum (Figure 4). The results revealed that 4 NtBPC genes formed collinear pairs with 4 SiBPC genes, while 4 NtBPC genes formed collinear pairs with 5 CaBPC genes (Figure 4). These results suggest that these genes may have originated before the diversification of Solanaceae species and retained their conserved functional roles.

3.3. Gene Structures and Amino Acid-Conserved Structures of NtBPCs

To elucidate the structural conservation and variability of the 12 NtBPC genes during evolution, we further analyzed their gene structures and conserved domains. As shown in Figure 5A, only NtBPC5, NtBPC7, and NtBPC8 contained untranslated regions (UTRs), while the remaining NtBPC genes lacked UTRs. Additionally, analysis of conserved amino acid motifs in these NtBPCs revealed that all NtBPCs retained the conserved GAGA-binding domain (Figure 5B). We also used the MEME program to explore the 10 most conserved motifs of NtBPC and annotated them using InterProScan (v5.68-99.0) (Supplementary Table S4). The results showed that NtBPC proteins within the same subfamily exhibited similar motif compositions, whereas those from different subfamilies displayed significant differences in motif compositions (Figure 5C). Specifically, proteins in Subfamily I contained motifs 1, 2, 4, 5, 6, 7, and 8; Subfamily II proteins contained motifs 1, 2, 3, 4, 5, 6, 7, and 9; and Subfamily III proteins contained motifs 1, 2, 4, 6, and 7 (Figure 5C).
Overall, the gene structures and conserved motifs of NtBPCs within the same subfamily strongly support the subfamily classification results obtained from phylogenetic analysis.

3.4. Cis-Elements Analysis of NtBPC Gene Promoters

To explore the potential roles and regulatory mechanisms of NtBPC genes, cis-element prediction was conducted for the 2000 bp promoter sequences upstream of the start codon of each NtBPC gene. A total of 944 cis-elements were identified, mainly associated with plant hormone responses, growth and development responses, stress responses, and light regulation in the promoters of NtBPC genes (Figure 6, Supplementary Table S5). Notably, light-responsive elements were the most prevalent among these cis-elements, present in the promoters of all NtBPC genes except NtBPC6, indicating that NtBPC genes might be regulated by light and subsequently participate in plant growth and development (Figure 6, Supplementary Table S5). Furthermore, cis-elements related to methyl jasmonate (JA) and abscisic acid (ABA) responses were more abundant than those associated with salicylic acid (SA), gibberellin (GA), and auxin responses (Figure 6, Supplementary Table S5). The promoters of NtBPC genes also contained various stress-responsive elements, including anaerobic induction elements, defense and stress response elements, and low-temperature response elements (Figure 6, Supplementary Table S5). Additionally, development-related elements such as meristem expression, zein metabolism regulation, circadian rhythm control, and endosperm expression were found in some NtBPC promoters. These results suggest that NtBPC genes may play important roles in light response, hormone response, stress response, and plant growth and development.

3.5. Expression Analysis of NtBPC Genes in Different Tissues

To further investigate the potential functions of each NtBPC gene, their expression levels were analyzed in the roots, stems, leaves, and shoot apical meristems of K326 (Figure 7). Due to the high similarity of the mRNA sequences of NtBPC genes, it is difficult to design sequence-specific primers for each copy. According to the mRNA sequence characteristics of these 12 NtBPC genes, we designed a pair of primers to detect the expression levels of NtBPC5 and NtBPC12, respectively. Additionally, five pairs of primers were designed for detecting the expression levels of NtBPC1 and NtBPC2, NtBPC3 and NtBPC4, NtBPC6 and NtBPC9, NtBPC7 and NtBPC8, as well as NtBPC10 and NtBPC11 (Supplementary Table S1). RT-qPCR results showed that all NtBPCs were expressed in roots, stems, leaves, and shoot apical meristems, with their expression levels in stems and leaves generally higher than those in roots and shoot apical meristems (Figure 7). Further comparative analysis demonstrated that NtBPC5 exhibited the highest expression levels in roots, stems, leaves, and shoot apical meristems among these 12 NtBPCs (Figure 7).
Overall, these results suggest that NtBPCs are capable of functioning on the roots, stems, leaves, and shoot apical meristems of tobacco, with more prominent functional roles in stems and leaves.

3.6. Expression Analysis of NtBPCs Under Different Stress Treatments

To investigate the function of NtBPC genes in response to abiotic stresses, we analyzed the expression levels of NtBPC genes under the treatments of drought, NaCl (200 mM), and cold (4 °C) using RT-qPCR. Our results showed that among the 12 NtBPC genes, the expression of NtBPC1/NtBPC2, NtBPC3/NtBPC4, and NtBPC7/NtBPC8 was induced exclusively by cold, NaCl, and drought treatments, respectively, compared with the control (CK) (Figure 8). The expression levels of NtBPC5 and NtBPC6/NtBPC9 were significantly increased by both cold and NaCl treatments, while those of NtBPC10/NtBPC11 were significantly elevated by both NaCl and drought treatments (Figure 8). Interestingly, the NtBPC12 expression was not induced by any of the cold, NaCl, or drought treatments. These results revealed the functional divergence among the NtBPC genes in response to abiotic stresses inclusive of cold, NaCl, and drought.

4. Discussion

Plant growth and development are precisely controlled by the complex regulatory networks constructed by integrating external environmental cues with internal signals, in which TFs serve as core regulatory participants [28,29]. BPCs, a family of plant-specific TFs, have been shown to play a crucial role in regulating plant growth, development, and responses to abiotic stresses in A. thaliana and other crops [5,12,13,30]. However, the basic information and function of BPC family members in tobacco have not been studied. Previous studies have shown that members of multigene families often exhibit substantial functional divergence, and that a systematic analysis of the sequence characteristics, evolutionary relationships, and expression patterns of each family member can provide valuable insights into their respective functions [31,32,33]. Therefore, we analyzed the number, gene structure, evolutionary relationship, expression patterns of NtBPCs, among other aspects.
In this study, a total of 12 NtBPC genes were identified from tobacco and categorized into three groups (Figure 3; Supplementary Table S2), which is consistent with the grouping number of BPC genes in A. thaliana, B. napus and C. sativus [5,16,19]. Among them, only five NtBPC genes were mapped to chromosomes, while the others were not assigned to specific chromosomes (Figure 1). This may be attributed to the large genome of tobacco and the fact that its sequencing has not yet been fully completed. We also found that 10 NtBPCs are localized in the nucleus (Figure 2; Supplementary Table S3), which is consistent with the fact that TFs usually function in the nucleus. Further analysis showed that NtBPC members within the same subfamily exhibit similar exon–intron structures and conserved motif compositions, indicating a closer evolutionary relationship among these members (Figure 5). Conversely, the members from different subfamilies of NtBPCs displayed distinct exon/intron and conserved motif compositions (Figure 5), implying functional diversification among the different subfamily members. Furthermore, all NtBPC proteins contain a conserved GAGA-binding domain that can bind to GA-rich box cis-elements [4,5], suggesting the potential functional conservation. Previous studies have demonstrated that AtBPC proteins exhibit both functional redundancy and antagonistic effects in regulating plant growth and development of A. thaliana. For instance, AtBPC1, AtBPC2, AtBPC4, and AtBPC6 can all regulate root development by directly repressing the expression of AtABI4 [11]. However, AtBPC3 acts antagonistically toward other AtBPC proteins in regulating the circadian clock and flowering time, with overexpression of AtBPC3 leading to growth defects similar to those seen in the higher-order bpc mutants (atbpc1 atbpc2 atbpc4 atbpc6) [10]. Therefore, the specific functions of NtBPCs need to be verified through genetic experiments.
The cis-elements in gene promoters are critical for their expression and function [34,35]. We found that the cis-acting elements involved in hormone responsiveness were widely present in the promoters of NtBPC genes such as auxin, GA and ABA (Figure 6, Supplementary Table S5). Studies have reported that the mutation of CsBPC2 exerts an adverse effect on salt-induced ABA biosynthesis and the expression of ABA signaling-related genes [36]. The apple MdBPC2 directly represses the expression of auxin biosynthesis genes MdYUCCA2a and MdYUCCA6b, and reduces auxin accumulation in MdBPC2 overexpression lines [37]. Additionally, previous studies also showed that ectopic overexpression of Camellia japonica BPC5 in A. thaliana results in the morphological defects of rosette leaves with fewer, smaller and upwardly curled leaves [38]. PtoBPC1 repressed PtoP4H9 expression and xylem cell expansion to improve poplar stem growth of populus [39]. AtBPC1, AtBPC2, AtBPC4 and AtBPC6 regulate root development by directly inhibiting the expression of AtABI4 [11]. Compared with the wild type, the root surface area, volume, and number of Csbpc2 mutants were significantly reduced, accompanied by a shift in root system architecture from dichotomous branching to herringbone branching [40]. Our results showed that NtBPCs were expressed in roots, stems, leaves, and shoot apical meristems (Figure 7). Notably, the expression levels of most NtBPCs genes in stems and leaves exceeded those in other tissues (Figure 7). Therefore, we infer that NtBPC genes may influence the growth and development of roots, stems, leaves, and shoot apical meristems, especially stems and leaves.
In addition, studies reported that seed germination of CsBPC2 overexpression lines was inhibited under the conditions of NaCl and PEG [16]. Compared to the wild type, the Csbpc2 mutants manifested enhanced leaf wilting, reduced biomass, as well as elevated levels of malondialdehyde and electrolytic leakage under salt stress conditions [36]. AtBPC1/AtBPC2 positively regulated plant salt tolerance by inhibiting GALACTAN SYNTHASE 1 expression and β-1,4-galactan accumulation [41]. These results demonstrate that plant BPC genes could play an important role in responding to abiotic stresses. We also identified low-temperature and drought stress response elements in the promoter regions of the NtBPC genes (Figure 6). Consistently, RT-qPCR results showed that except for NtBPC12, the expression of other NtBPC genes could be induced by cold, drought or salt stress (Figure 8). Notably, the expression of NtBPC1/NtBPC2, NtBPC3/NtBPC4, and NtBPC7/NtBPC8 was only upregulated by cold, NaCl, and drought treatments, respectively (Figure 8). The expression of NtBPC5 and NtBPC6/NtBPC9 were significantly induced by both cold and NaCl treatments, while those of NtBPC10/NtBPC11 were significantly elevated by both NaCl and drought treatments (Figure 8). Accordingly, these findings preliminarily imply that NtBPC genes play a crucial role in determining the tobacco’s tolerance to cold, NaCl, and drought stresses, but their functions are differentiated. Interestingly, the NtBPC12 gene did not exhibit significant expression changes under the cold, NaCl, and drought treatments (Figure 8), suggesting that this gene may not participate in the stress response process of tobacco. However, the specific functions of NtBPC genes in response to cold, NaCl, and drought stresses need further investigation.

5. Conclusions

In this study, a total of 12 NtBPC genes were identified from the genome of tobacco K326, which belong to three subfamilies and exhibit high similarity in terms of structural composition and the distribution of conserved motifs. Further analysis revealed that their expression patterns vary across different tissues and under drought, cold, and salt treatments. This study provides a basis for analyzing the function of NtBPC genes in tobacco. In the future, bioinformatics and molecular biology technologies can be combined to systematically explore the functions of NtBPC genes in tobacco growth and development as well as in response to abiotic stresses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092084/s1, Table S1: Primers used in this study; Table S2: List of NtBPC sequences; Table S3: The physicochemical properties of 12 NtBPCs; Table S4: Sequences of 10 predicted motifs of NtBPC proteins; Table S5: Detailed information of cis-acting elements identified in promoter region of NtBPCs.

Author Contributions

Study conception, design, and manuscript revision: Y.Z. and Z.L.; data collection: S.W. and S.J.; analysis and interpretation of results: Y.Z., S.J. and X.J.; draft manuscript preparation: Y.Z. and S.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation in Shaanxi Province of China (grant no. 2023-JC-QN-0194); the Scientific Research Fund Projects of Yangling Vocational & Technical College in 2022 (grant no. ZK22-57); the Forestry Scientific Research Project of the Modern Forestry Vocational Education Group in Northern China (grant no. LZJB2024KY008).

Data Availability Statement

The data presented in this study are available in the article and the Supplementary Materials. For further inquiries, you can contact the corresponding author directly.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Chromosomal distribution of NtBPC genes in tobacco. The scale bar on the left side indicates the size of the chromosome. The relative position of each NtBPC gene is indicated by a black line.
Figure 1. Chromosomal distribution of NtBPC genes in tobacco. The scale bar on the left side indicates the size of the chromosome. The relative position of each NtBPC gene is indicated by a black line.
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Figure 2. Subcellular localization of NtBPC proteins fused with GFP in N. benthamiana leaf epidermal cells. GFP, a green fluorescent protein. mCherry, a nuclear-localized protein fused with a red fluorescent protein. Merge, merged image of GFP, mCherry, and bright field images (Scale bar represents 20 μm).
Figure 2. Subcellular localization of NtBPC proteins fused with GFP in N. benthamiana leaf epidermal cells. GFP, a green fluorescent protein. mCherry, a nuclear-localized protein fused with a red fluorescent protein. Merge, merged image of GFP, mCherry, and bright field images (Scale bar represents 20 μm).
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Figure 3. Phylogenetic analysis of BPC family proteins from Arabidopsis thaliana and Nicotiana tabacum. Blue circles indicate AtBPC proteins, red stars indicate NtBPC proteins. Bootstrap values are indicated by red and black circles on the nodes. The neighbor-joining method, with parameters set as Poisson correction, pairwise deletion, and 1000 bootstrap replicates, was employed to construct the phylogenetic tree of BPC proteins using MEGA X.
Figure 3. Phylogenetic analysis of BPC family proteins from Arabidopsis thaliana and Nicotiana tabacum. Blue circles indicate AtBPC proteins, red stars indicate NtBPC proteins. Bootstrap values are indicated by red and black circles on the nodes. The neighbor-joining method, with parameters set as Poisson correction, pairwise deletion, and 1000 bootstrap replicates, was employed to construct the phylogenetic tree of BPC proteins using MEGA X.
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Figure 4. Synteny analysis of BPC genes among Capsicum annuum, Solanum lycopersicum and Nicotiana tabacum. Gray lines indicate all collinear blocks within C. annuum, S. lycopersicum and N. tabacum, while the blue lines depict the orthologous relationships of BPC genes among C. annuum, S. lycopersicum and N. tabacum.
Figure 4. Synteny analysis of BPC genes among Capsicum annuum, Solanum lycopersicum and Nicotiana tabacum. Gray lines indicate all collinear blocks within C. annuum, S. lycopersicum and N. tabacum, while the blue lines depict the orthologous relationships of BPC genes among C. annuum, S. lycopersicum and N. tabacum.
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Figure 5. Schematic diagram of gene structures (A), conserved domains (B), and conserved motifs (C) of NtBPC genes. In (A), green boxes represent exons, and light gray lines indicate introns. UTRs, the untranslated regions. CDS, coding sequence. The scale at the bottom represents the length of genes (A) and proteins (B,C).
Figure 5. Schematic diagram of gene structures (A), conserved domains (B), and conserved motifs (C) of NtBPC genes. In (A), green boxes represent exons, and light gray lines indicate introns. UTRs, the untranslated regions. CDS, coding sequence. The scale at the bottom represents the length of genes (A) and proteins (B,C).
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Figure 6. Predicted cis-elements in the promoters of NtBPC genes. The 2000 bp promoter sequence upstream of the start codon of each NtBPC gene was analyzed by PlantCARE.
Figure 6. Predicted cis-elements in the promoters of NtBPC genes. The 2000 bp promoter sequence upstream of the start codon of each NtBPC gene was analyzed by PlantCARE.
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Figure 7. Expression levels of NtBPC genes in different tobacco tissues. Relative expression values were calculated by normalizing against NtHSC70-1 with the 2−ΔΔCT method. Data are means ± SD from three biological replicates.
Figure 7. Expression levels of NtBPC genes in different tobacco tissues. Relative expression values were calculated by normalizing against NtHSC70-1 with the 2−ΔΔCT method. Data are means ± SD from three biological replicates.
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Figure 8. Expression levels of 12 NtBPCs in leaves of tobacco under the treatment of drought, NaCl (200 mM), and cold (4 °C). Relative expression values were calculated by normalizing against NtHSC70-1 with the 2−ΔΔCT method. Data are means ± SD from three biological replicates. Asterisks denote significant differences between the control (CK) and treatment groups (two-tailed paired Student’s t-test, * p < 0.05, *** p < 0.001, ns p ≥ 0.05).
Figure 8. Expression levels of 12 NtBPCs in leaves of tobacco under the treatment of drought, NaCl (200 mM), and cold (4 °C). Relative expression values were calculated by normalizing against NtHSC70-1 with the 2−ΔΔCT method. Data are means ± SD from three biological replicates. Asterisks denote significant differences between the control (CK) and treatment groups (two-tailed paired Student’s t-test, * p < 0.05, *** p < 0.001, ns p ≥ 0.05).
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Zhang, Y.; Jia, S.; Wang, S.; Ji, X.; Liu, Z. Genome-Wide Identification and Characterization of BPC Transcription Factors in Tobacco (Nicotiana tabacum). Agronomy 2025, 15, 2084. https://doi.org/10.3390/agronomy15092084

AMA Style

Zhang Y, Jia S, Wang S, Ji X, Liu Z. Genome-Wide Identification and Characterization of BPC Transcription Factors in Tobacco (Nicotiana tabacum). Agronomy. 2025; 15(9):2084. https://doi.org/10.3390/agronomy15092084

Chicago/Turabian Style

Zhang, Ying, Shengwu Jia, Sisi Wang, Xiaolian Ji, and Zijin Liu. 2025. "Genome-Wide Identification and Characterization of BPC Transcription Factors in Tobacco (Nicotiana tabacum)" Agronomy 15, no. 9: 2084. https://doi.org/10.3390/agronomy15092084

APA Style

Zhang, Y., Jia, S., Wang, S., Ji, X., & Liu, Z. (2025). Genome-Wide Identification and Characterization of BPC Transcription Factors in Tobacco (Nicotiana tabacum). Agronomy, 15(9), 2084. https://doi.org/10.3390/agronomy15092084

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