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Genome-wide identification and expression analysis of the bZIP transcription factor family genes in response to abiotic stress in Nicotiana tabacum L.
BMC Genomics volume 23, Article number: 318 (2022)
The basic leucine zipper (bZIP) transcription factor (TF) is one of the largest families of transcription factors (TFs). It is widely distributed and highly conserved in animals, plants, and microorganisms. Previous studies have shown that the bZIP TF family is involved in plant growth, development, and stress responses. The bZIP family has been studied in many plants; however, there is little research on the bZIP gene family in tobacco.
In this study, 77 bZIPs were identified in tobacco and named NtbZIP01 through to NtbZIP77. These 77 genes were then divided into eleven subfamilies according to their homology with Arabidopsis thaliana. NtbZIPs were unevenly distributed across twenty-two tobacco chromosomes, and we found sixteen pairs of segmental duplication. We further studied the collinearity between these genes and related genes of six other species. Quantitative real-time polymerase chain reaction analysis identified that expression patterns of bZIPs differed, including in different organs and under various abiotic stresses. NtbZIP49 might be important in the development of flowers and fruits; NtbZIP18 might be an important regulator in abiotic stress.
In this study, the structures and functions of the bZIP family in tobacco were systematically explored. Many bZIPs may play vital roles in the regulation of organ development, growth, and responses to abiotic stresses. This research has great significance for the functional characterisation of the tobacco bZIP family and our understanding of the bZIP family in higher plants.
Transcription factors (TFs) are proteins that combine with specific short sequences of DNA to regulate the expression of genes [1, 2]. Important regulatory factors, TFs bind to the promoter regions of target genes and activate or inhibit their transcription. Among the TF families, the basic leucine zipper (bZIP) TF family is the most widely distributed and conserved in the domain of eukaryotes, found in humans, plants, animals, and microorganisms [3, 4].
The bZIP TF family is named after the bZIP domain . The bZIP protein sequence contains 2 conserved bZIP domains with a total length of ~ 60–80 amino acids. The first is a highly conserved domain containing an N- × 7-R/K sequence, which consists of approximately 16 amino acid residues and functions in localization and specific binding to DNA [6, 7]. The second is a relatively poorly conserved leucine zipper region characterised by leucine in the last position of every seven amino acids, and hydrophobic residues at positions 3 and 4. The α-helix is a necessary structure of dimer required for bZIP proteins to bind to DNA [8,9,10]. The core sequence of bZIP TFs is the cis acting element of ACGT, e.g., as found in the G-box (CACGTG), C-box (GACGTC), the abscisic acid (ABA)-responsive element, and the A-box (ACGTA) [11,12,13,14].
Research into the bZIP family in different species will bring new insights into its evolution and function. Previous studies have shown that the bZIP TF family is involved in the regulation of genes involved in organ and tissue differentiation [15,16,17,18], seed storage [19, 20], pathogen defence , the unfolded protein response [22,23,24], photomorphogenesis and light signal regulation [25, 26], hormone and sugar signaling processes [27, 28], and responses to abiotic stress [3, 29, 30]. Different subfamilies regulate different biological processes. Studies have shown that AtbZIP30 is involved in regulating cell development, organ development, hormone homeostasis, and meristematic processes . Some bZIP TFs are activated under abiotic stress; they can combine with the promoter regions of key genes in signaling pathways to regulate their transcription levels and, in turn, plant resistance to stress. For example, research has identified that the expression of EcbZIP60, a gene homologous to AtbZIP60, is upregulated in response to drought and salt stresses . In Arabidopsis thaliana, HY5, a member of the bZIP TF family, has been shown to inhibit hypocotyl growth and lateral root development, and promote pigment accumulation in a light-dependent manner . OsbZIP12 is a member of the bZIP family in rice that has been shown to be involved in ABA signal transduction and regulation of drought resistance . RsbZIP031 and RsbZIP059 in radish were significantly downregulated under high temperature and salt stress, respectively . In pineapple root tissues, AcobZIP24 has a significant response to salt, drought, cold, and heat stresses at 24 and 48 h . These examples illustrate the response of bZIP TFs in plants to various abiotic stresses.
Nicotiana tabacum L. is a tobacco plant of the Solanaceae. Tobacco is an important cash crop in agriculture, and is widely cultivated in more than 120 countries around the world [31,32,33,34]. Besides A. thaliana, tobacco is also often used as a model plant for the study of biological mechanisms because of the advantages conferred by its short growth time, disease susceptibility, and easy genetic transformation . In addition, there is new interest in tobacco for its potential in biopharmaceuticals and biofuel production [36, 37]. Thriving in the natural environment, tobacco is stressed by salt, drought, cold, heat, and flooding, which seriously impact its growth, yield, and quality [17, 18, 38,39,40]. The bZIP family has been comprehensively identified and studied, with 75 bZIPs identified in A. thaliana [7, 8], 89 in rice , 131 in soybean , 55 in grape , 90 in potato , and 52 in Carthamus tinctorius , with further bZIP genes identified in other plant species [44,45,46]. However, research into the bZIP family of tobacco is lacking. The development of the Tobacco Genome Initiative has provided many opportunities for the study of genome-wide gene families in tobacco [19, 20]. Furthermore, the emergence of next-generation sequencing technology enabled the problems of high cost and low yield to be overcome, making the development of tobacco genome sequencing more feasible [8, 9, 47,48,49]. In this study, we identified bZIPs from Nicotiana tabacum L., and studied their gene structure, chromosomal location, duplication, gene expression patterns, and responses to 6 abiotic stresses. We also studied the changes in enzyme activity occurring in plants under stress. In addition, an analysis of the homology between bZIPs in A. thaliana, Oryza sativa, Fagopyrum tataricum, Solanum lycopersicum, Hordeum vulgare, Vitis vinifera, Solanum tuberosum and tobacco was conducted. This study provides a theoretical basis for the mechanisms of stress resistance in tobacco and other species, and could open new research avenues in the breeding of high-quality tobacco varieties.
Identification of NtbZIPs genes in tobacco
In this study, we identified 77 NtbZIPs in the tobacco database using two BLAST methods, and named them NtbZIP01 through to NtbZIP77 according to their chromosomal locations (from top to bottom) (Additional file 1: Table S1). The physical properties and basic characteristics of the predicted 77 NtbZIPs were analysed, including the coding sequence length (CDS), protein molecular weight (Mw), isoelectric point (pI), and predicted subcellular location (W) (Additional file 1: Table S1). The protein length of the predicted 77 NtbZIPs ranged from 85 (NtbZIP43) to 973 amino acids (NtbZIP76), with an average of 369 amino acids. The lowest Mw was 10.11 kDa (NtbZIP43), whilst the highest was 104.64 kDa (NtbZIP76); the average Mw was 40.77 kDa. The pI ranged from 4.17 (NtbZIP32) to 10.19 (NtbZIP52), with a mean of 7.11. Furthermore, analysis of predicted subcellular locations indicated that 64 NtbZIPs were located to the nuclear, 8 to chloroplasts, 3 (NtbZIP21, NtbZIP43, NtbZIP68) to the cytoplasm, 1 (NtbZIP02) to vacuole, and 1 (NtbZIP51) to the endoplasmic reticulum. The ratio of NtbZIPs to total genes in the tobacco genome was approximately 0.10%, similar to Chinese jujube (0.13%) , but lower than Chenopodium quinoa (0.16%) , castor bean (0.19%) , A. thaliana (0.20%) , and banana (0.35%) .
Phylogenetic analysis and classification of bZIPs in tobacco
To study the evolutionary relationship between the bZIP families of A. thaliana and tobacco, a phylogenetic tree was constructed based on the amino acid sequences of bZIPs in A. thaliana and tobacco (Fig. 1; Additional file 1: Table S1). We used the method proposed by Jakoby et al.  to construct an unrooted neighbour-joining tree for classification. The 77 identified NtbZIPs were clustered into 11 subfamilies (A-G, I, J, K, S) and four ‘orphan genes’. Among these 11 subfamilies, 9 (A-G, I, and S) were similar to those found in A. thaliana, and the subfamilies were named to be consistent with A. thaliana . We also identified 2 new subfamilies, J and K. These results indicated that the bZIP family is conserved in the evolutionary history of tobacco; these conserved subfamilies may play an important role in the process of evolution. The A subfamily was the largest subfamily identified, with 14 NtbZIP members; the S subfamily was second largest, containing 13 NtbZIPs. The smallest subfamilies were the B and D subfamilies, each containing 2 genes. The C, E, F, G, and I subfamilies had 3, 7, 5, 7, and 10 members, respectively. Subfamily H from A. thaliana (consisting of only AtbZIP56 and AtbZIP64), contained no NtbZIP members, indicating that this group was lost during tobacco evolution (Fig. 1). Conversely, subfamilies J and K contained only NtbZIPs (6 and 4, respectively), suggesting that this may be a unique evolutionary direction in tobacco. Aside from the 11 subfamilies, 4 genes (NtbZIP20, NtbZIP23, NtbZIP25, and NtbZIP56) were not divided into any subfamily, which suggested that these 4 genes may have unique functions. The phylogenetic trees of A. thaliana and tobacco identified some NtbZIPs that were closely linked to AtbZIPs; these genes could potentially be homologous with the AtbZIPs and have similar physiological functions.
Gene structure and motif analysis of the NtbZIP genes
By comparing the corresponding genomic DNA sequences, all NtbZIPs were further analysed to better understand the structural characteristics, including the number and sistruction of exons and introns (Fig. 2a,b). The numbers of introns present in the NtbZIPs ranged from 0 to 17 (Additional files 1: Tables S1). In total, 13 NtbZIP genes (16.88%) had no intron, 8 (10.39%) had 1 intron, 6 (7.79%) had 2 introns, 22 (28.57%) had 3 introns, 4 (5.19%) had 4 introns, 2 (2.60%) had 5 introns, 1 (1.30%) had 6 introns, 3 (3.90%) had 7 introns, 1 (1.30%) had 8 introns, 4 (5.19%) had 11 introns, 1 (1.30%) had 16 introns, 5 (6.49%) had 17 introns, respectively. The genes with no intron belonged to the A, F, and S subfamilies, and predominantly in the S subfamily. The 22 NtbZIPs with 3 introns accounted for the largest proportion of genes (28.57%). NtbZIP24, 42, 48, 63, and 68 had the most number of introns, 17, and all of these genes belonged to the J subfamily. In general, genes in the same subgroup had a similar structure. For example, all genes in the E subfamily (except NtbZIP40) had 4 exons and 3 introns; all genes in the J subfamily (except NtbZIP55) had 18 exons and 17 introns.
In order to study the characteristic regions of the NtbZIP proteins, we used the MEME online tool to predict conserved motifs. A total of 10 conserved motifs (motifs 1–10) were identified from the 77 NtbZIP proteins (Fig. 2c, Additional file 2: S2). The length of the motifs ranged from 15 (motif 3) to 50 (motif 2, 4, 7, 8, 9, 10) (Additional file 2: S2). Motif 1 and 3 were widely distributed in the majority of NtbZIPs and these 2 motifs were very closely positioned in proteins, suggesting that the two motifs may play an important role in particular functions. As expected, members from the same subfamily shared similar motif compositions, particularly between a few close members. For example, members of the J subfamily had the same motifs: 3, 4, 6, 7, and 8. In subfamily S, apart from NtbZIP43 which only contained motif 3, the rest of the members included both motifs 1 and 3; in addition, motif 9 was found in all the members of subfamily F. Furthermore, we also found that some motifs only appeared in certain subfamilies; e.g., motif 8 only appeared in subfamily J, indicating that this motif is specific to this subfamily. Analysis also identified that some of the 10 motifs only occurred in particular orders. In the J subfamily, except for where motifs were repeated, the motifs only appeared in a specific order: 6–3-4–7-8. Similarly, this also occurred in the F subfamily, in which the order of motifs was 9–1-3–10. In summary, the genes that in the same subfamily shared similar gene structure ana motif composition, providing further support for the phylogenetic analysis and subfamily classifications.
Chromosome location and duplication of bZIPs in tobacco
On the basis of the tobacco genome sequence, we constructed a map of the physical positions of the NtbZIPs (Fig. 3). As shown in Fig. 3, all the identified 77 NtbZIPs were located on tobacco chromosomes, and the nomenclature of the genes was guided by their physical positions on tobacco Chr1 to Chr24. Aside from Chr8 and Chr14, on which no NtbZIP was distributed, the number of genes distributed on the remaining chromosomes ranged from 1 to 15. Chr17 contained the largest number of NtbZIPs (15 genes, 19.48%), followed by Chr24 (8 genes, 10.39%), Chr19 (7 genes, 9.09%) and Chr5 (6 genes, 7.79%). Chr1, Chr3, Chr7, Chr11, Chr13, Chr16, Chr18, Chr20, and Chr21 included just 1 gene (~ 1.30%), Chr22 contained 2 NtbZIPs (~ 2.60%), and Chr2, Chr9, Chr15, and Chr23 each contained 3 NtbZIPs (~ 3.90%). Chr6 and Chr10 had 4 (~ 5.19%) NtbZIPs, whilst Chr4 and Chr12 all included 5 NtbZIPs (6.49%). There was a correlation between chromosome size and the number of genes on chromosome. Such as, the largest chromosome (Chr17) contains 15 genes, and the smallest chromosome (Chr21) contains only one gene.
Furthermore, 16 pairs of segmental duplication were observed in the NtbZIPs (Fig. 4, Additional file 3: Table S3) and no tandem duplication was observed. Twenty-nine (37.66%) paralogs were identified in the NtbZIP family, indicating an evolutionary relationship between these bZIP members (Fig. 4). The paralogous NtbZIPs were unevenly distributed between 13 tobacco linkage groups (LGs) (Fig. 4). LG17 contained the most NtbZIPs (9), whilst LG7, LG9, LG10, LG12, LG18, and LG21 had the least (1). LG2, LG4, LG5, and LG6 each contained 2 genes, and there were 3 genes in LG19 and LG24. We also found that the 16 pairs of paired genes were linked within their subfamilies of S, A, F, G, and I.
Synteny analysis of NtbZIPs
To analyse the evolutionary relationship of the tobacco bZIP family, we constructed 7 synteny maps, comparing tobacco with six representative species, including A. thaliana, S. lycopersicum, F. tataricum, O. sativa, H. vulgare, V. vinifera and S.tuberosum (Fig. 5, Additional file 4: Table S4). In total, 68 NtbZIPs displayed a syntenic relationship across the 7 species, indicating that the tobacco bZIPs were highly conserved on the corresponding chromosomes in the process of evolution. The largest number of NtbZIPs displaying a syntenic relationship occurred when comparing the tobacco genes to S.tuberosum (60),, followed by S. lycopersicum (59), V. vinifera (45), A. thaliana (29), F. tataricum (27), O. sativa (9), and lastly H. vulgare (3) (Additional file 4: Table S4). The number of orthologous pairs between the other 7 species analysed (A. thaliana, O. sativa, F. tataricum, S. lycopersicum, H. vulgare, V. vinifera and S.tuberosum) was 44, 14, 35, 97, 4, 59 and 92, respectively. These result illustrated that the genetic relationship of NtbZIP genes, SlbZIP genes and StbZIP genes were close, which was reasonable because they are all Solanaceae plants. Several NtbZIPs were associated with at least 6 syntenic gene pairs: NtbZIP07, NtbZIP21, NtbZIP24, NtbZIP30, NtbZIP33, NtbZIP38, NtbZIP41, NtbZIP43, NtbZIP49, NtbZIP50, NtbZIP52, NtbZIP57, NtbZIP59, NtbZIP60, and NtbZIP73, indicating that these genes may have played an important role in the evolution of the bZIP family in tobacco. Furthermore, NtbZIP33 was found in tobacco and all of the other 7 species, indicating that this orthologous pair appeared earlier than ancestral differentiation, and that NtbZIP33 may have played a vital role in the evolution of the tobacco bZIP family.
Expression patterns of NtbZIPs in different plant tissues and organs
To investigate the physiological roles of the NtbZIPs, we selected 11 NtbZIPs from different subfamilies to study gene expression patterns. The expression levels of these NtbZIPs in eight tissues (calyx, petal, anther, stigma, leaf, stem, fruit, and root) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) (Fig. 6a). The different expression patterns found for each gene in different organs indicate that NtbZIPs have different functions in tobacco. Most of the genes were expressed in all organs, except NtbZIP04, which was not expressed in the leaf or stem. NtbZIP69 genes was more highly expressed in the calyx than in other organs. NtbZIP29 and NtbZIP69 were highly expressed in petal tissue, and NtbZIP70 was highly expressed in anther tissue. NtbZIP01 was more highly expressed in both stigma and leaf tissue than in other tissues, and its expression in these tissues was higher than other NtbZIPs; similarly NtbZIP49 was more highly expressed in stem, fruit, and root tissue than other genes. In addition, we analysed whether the expression patterns of the 11 NtbZIPs in the 8 organs were correlated (Fig. 6b). This analysis found that the expression of different genes in the plant organs were significantly correlated; some genes were positively correlated (NtbZIP49 with NtbZIP37, NtbZIP69, and NtbZIP70; NtbZIP01 with NtbZIP04, NtbZIP76, and NtbZIP18), whilst some were negatively correlated (NtbZIP37 and NtbZIP29; NtbZIP70 with NtbZIP01, NtbZIP18, NtbZIP29, NtbZIP76). All of the 11 NtbZIPs showed positive correlation with different tissues (Additional file 5: Figure S1). For example, the NtbZIP70, NtbZIP74, NtbZIP29 showed highly positive correlation with the anthers, the stem and the petals, respectively. However, NtbZIP63 showed negative correlation with anthers.
Expression patterns of NtbZIPs in response to different stresses
To study the expression of NtbZIPs under abiotic stress, we used qRT-PCR to analyse the expression of 11 NtbZIPs in roots, stems, and leaves under 6 abiotic stresses: polyethylene glycol (PEG), flooding, heat, cold, NaCl, and strong ultraviolet (UV) radiation treatments (Fig. 7a). The expression patterns of NtbZIPs differed in response to the 6 abiotic stresses, with some NtbZIPs either significantly induced or inhibited. In the early stages of stress treatments, the expression patterns of most genes changed significantly (Fig. 7a); in response to different stress treatments, the expression of some NtbZIPs also changed with time or in different organs. For example, in response to PEG stress, NtbZIP04, NtbZIP18, and NtbZIP49 were first significantly upregulated in root tissue, and then downregulated; NtbZIP49 also demonstrated this pattern of expression in stem and leaf tissue. In response to heat stress, NtbZIP18 and NtbZIP29 were significantly downregulated in the root, stem, and leaf, whereas NtbZIP74 was first significantly upregulated, and then downregulated in these tissues. Some other genes showed changes in specific organs: for example, the response of NtbZIP76 to cold stress was significant only in the stem. In addition, we analysed whether there were correlations between the expression patterns of these NtbZIPs (Fig. 7b), detecting positive correlations among most NtbZIPs. NtbZIP49 and NtbZIP63 were significantly positively correlated, and 4 genes (NtbZIP04, NtbZIP18, NtbZIP37 and NtbZIP49) were significantly positively correlated with NtbZIP29. Under different abiotic stresses, plant tissues showed different correlations with gene expression patterns (Additional file 6: Figure S2). Under flooding stress, leaf tissue showed significant positive correlations with NtbZIP69, but a negetive correlation with NtbZIP29. Similarly, under heat stress, root tissue was significantly positively correlated with NtbZIP29, NtbZIP18 and NtbZIP01, but significantly negatively correlated with NtbZIP04 and NtbZIP76. Furthermore, The number of genes that positively related to stem tissue were more than that of leaf and root tissue under 6 abiotic stresses.
Tobacco is an important commercial crop and model plant. The bZIP family is one of the most important TF families found in the plant kingdom. Increasing evidence suggests that bZIP TFs play important roles in regulating growth and development, and responses to biotic and abiotic stresses [3, 15, 52]. Previous research on the functions of bZIP TFs has mainly focused on A. thaliana, and there were also relevant studies in other crops, vegetables, and fruits [4, 8, 9, 28, 42, 53]. Compared with other important plants, there has been little research on the bZIP family in tobacco; therefore, we systematically analysed the bZIP family in tobacco.
In this study, 77 bZIPs were identified from tobacco (3734 Mb) on the basis of a constructed phylogenetic tree. Previous studies have identified 75 bZIPs in A. thaliana (117 Mb) , 89 in rice (466 Mb) , 125 in maize (2182 Mb) [54, 55], 160 in soybean  (915 Mb), 55 in grape  (490 Mb), and 96 in tartary buckwheat  (489 Mb). Results from this study suggest that the number of bZIPs in tobacco is similar to that found in A. thaliana, more than in grape and in Carthamus tinctorius, and fewer than in rice and soybean. There is no correlation between the number of bZIPs in a species and genome size. According to cluster analysis and the similarity of protein sequences with A. thaliana, the 77 NtbZIPs divided into 11 subfamilies, and 2 subfamilies (J and K) were newly identified. The number of subfamilies was consist with the buckwheat , caster bean , more than A. thaliana  and grape , lower than Chinese jujube  and poplar . The J subfamily is consistent with grapevine  and Chinese jujube , whereas the K subfamily is the same as that in soybean  and radish . Subfamilies A and S had the largest number of bZIPs, similar to as seen in A. thaliana . Four of the NtbZIPs did not have obvious clusters; this also occurred in grape , and banana . The H subfamily did not contain any tobacco genes, which suggested that the H subfamily was lost in tobacco.
Gene replication, including tandem, segment, and whole genome replication, plays an important role in the evolution of organisms and the expansion of gene family . In this study, 16 pairs of segmental duplications were observed in the NtbZIP genes, without any tandem replications, which also occurred in poplar . These results suggest that segmental replication may contribute more than tandem duplications to the expansion of the bZIP family in tobacco, consistent with previous studies in other species, including rice , maize , grape , and cucumber . The number of bZIPs in tobacco is similar to that found in A. thaliana , more than in grape  and in Carthamus tinctorius  and fewer than in rice  and soybean . This difference may be caused by the whole-genome duplication event that occurred after the earliest ancestors of the other species diverged . These segment duplication events may be promote the occurrence and evolution of some NtbZIP genes, which also occurred in Populus trichocarpa  and Sorghum bicolor . From the results of chromosome distribution, there were no NtbZIP genes on Chr8 and Chr14, illustrating that the NtbZIP gene family may be affected by gene deletion during the process of evolution . Therefore, some bZIP genes may be caused by some replication events, which further illustrates that replication events play an important role in the rapid expansion of bZIP family members in plants.
In addition, the phylogenetic analysis was confirmed by gene structure and conserved motif analysis of the 77 bZIPs. Analysis of the gene structure of the NtbZIP family identified several differences among the NtbZIPs. We determined that the number of introns present varied from 0 to 17, with 16.88% of the genes containing no introns, similar to as seen in rice , soybean , and sorghum . The two duplicated genes tended to cluster in a subfamily, and previous studies have also shown such results . The results of phylogenetic analysis confirmed this, with NtbZIP30 and NtbZIP77 (1 exon and 0 introns), NtbZIP11 and NtbZIP75 (3 exons and 2 introns), and NtbZIP21 and NtbZIP59 (2 exons and 1 intron) clustered to subfamilies S, A, and F, respectively. The subfamilies G, J, and K contained genes with more than 9 introns, while other subfamilies had genes with fewer introns. Nuruzzaman et al.  studied the bZIP TF family in rice, and found that the rate of intron acquisition was slower than the rate of intron deletion after segmental duplication. Therefore, we can infer that the G, J, and K subfamilies may contain primitive genes. In most gene families, the increase and loss of introns and exons is one of the mechanisms for diversification , and this was also observed here. For example, NtbZIP47 had 1 intron and 2 exons, while its paralogous gene NtbZIP45 contained 0 introns and 1 exon, indicating the loss of an exon during evolution. A similar phenomenon also occurred in the bZIP gene families of apple  and soybean . The increase of introns is conducive to the adaptation of plants to the environment, which is beneficial to evolution. However, genes with fewer introns may be conducive to rapid response to the environment . In this study, a total of 10 motifs were identified in the 77 NtbZIP proteins. Although different subfamilies have different motifs and the motif encoding bZIP is conservative. Most members contained the motif 1 conserved domain, which contains an N- × 7-R/K conserved sequence, indicating conservation of the bZIP TF family in tobacco. Furthermore, analysis of the conserved motifs indicated that each subfamily member shared similar motifs, which were the same as those in buckwheat [27, 44]. For example, all NtbZIP proteins shares motifs 1, 3 and 5 in group I. Both Group S and I contain motifs 1 and 3. These results not only support the classification of the subfamilies, but also indicate that members in the same subfamily have similar evolutionary histories and physiological functions .
The bZIP family plays an important regulatory role in growth and development, including the development of flowers and fruits [51, 69, 70]. Analysis of gene expression profiles can provide an important basis for understanding the potential biological functions of genes. The functions of bZIP TFs in tobacco still need to be further studied. Here, we selected 11 NtbZIPs, clustered in different subfamilies and with significant differences, to study the expression patterns in different organs. All of the NtbZIP genes investigated were expressed in at least 1 of the organs tested, indicating that they may play a broad role in the growth and development of tobacco, similar to as the former reported research in soybean  and buckwheat . Most genes were expressed in all organs, consistent with previous studies [8, 43, 45, 72], however there were some genes that were not expressed in some of the organs, as previously seen in studies of grape, soybean, and apple [53, 56, 67]. For instance, the NtbZIP04 gene was not expressed in leaf and stem. In this study, according to the phylogenetic tree constructed by NtbZIP genes homologous to AtbZIP genes (Fig. 1), the function of NtbZIP gene homologous to AtbZIP can be predicted. Clarifying the function of these NtbZIP genes can lay a foundation for the growth and development of tobacco and other species in future. As the reproductive organs of plants, flowers and fruits are the main structures of all angiosperms . In comparison to non-reproductive tissues (root, stem, and leaf), the expression levels of NtbZIP29 was significantly higher in the petal, stigma and anther tissues. AtbZIP18, a gene in A. thaliana homologous to the tobacco gene NtbZIP29. The study of AtbZIP18 gene in A. thaliana showed that AtbZIP18 had high expression in pollen and played an important role in male gametophyte . Therefore, we speculated that NtbZIP29 is involved in the development of flowers and fruits. NtbZIP01 showed high expression in leaves. AtbZIP41, a gene homologous to NtbZIP01, has been proven to recognise and specifically bind with the G-box to form the G-box-binding factor 1/bZIP41 binding factor, which is constitutively expressed in A. thaliana leaves [74,75,76]. According to the subfamily classification result (Fig. 1), NtbZIP01 belongs to G subfamily that contains the G-box-binding factor. The specific expression of NtbZIP01 gene in leaf may be due to its special structure. The expression level of NtbZIP04 was highest in stigma; the A. thaliana genes homologous to NtbZIP04, AtbZIP34 and AtbZIP61, have been confirmed in several studies to interact with other bZIP TFs to participate in plant development [48, 74, 77]. The NtbZIP04 gene may be has similar function with the homologous genes of AtbZIP34 and AtbZIP61.
bZIP genes play an vital role in plant stress response, which have illustrated in previous studies [4, 8, 9]. We also studied the gene expression patterns of these 11 NtbZIPs under 6 abiotic stresses. The results showed that the expression of most of the NtbZIPs were significantly different under abiotic stresses, which was consistent with previous studies [28, 53, 78]. For example, under drought stress, NtbZIPs were upregulated in the root (9), stem (9), and leaf (7). There were 8, 5, 11 NtbZIPs upregulated in the root, stem, and leaf, respectively. Additionally, the same gene showed opposite expression patterns under different stress. NtbZIP04 gene was up-regulated in leaves under heat stress, but down-regulated under cold stress. These results illustrate that bZIP genes are widely involved in the response to abiotic stress and bZIP TFs participate in a complex cross regulatory network . NtbZIP49 and NtbZIP76 responded to PEG, flooding, heat, cold, and NaCl abiotic stresses at the same time, indicating that these two genes are co-expressesed under a variety of adverse conditions, which was consistent with the study of Sorghum bicolor . We found high expression of NtbZIP18 under all 6 abiotic stresses. NtbZIP18 is homologous with subfamily F members AtbZIP19, AtbZIP23, and AtbZIP24 of A. thaliana. Previous studies showed that AtbZIP24 is an important regulator of the salt stress response in plants, and regulates complex transcription networks involved in abiotic stress resistance [79, 80]. The NtbZIP18 gene may have similar functions to AtbZIP24 gene. Subsequent studies can verify the function of NtbZIP18 gene to prove whether it can be used to improve the resistance of varieties to various abiotic stress. NtbZIP49 showed the highest expression levels in stem tissue under NaCl stress; this gene is homologous with AtbZIP75 of A. thaliana, reported to respond to salt stress . Among the six abiotic stress, NtbZIP74 showed high expression in root. NtbZIP74 gene and AtbZIP67 are homologous genes and both come from subfamily A that is mainly related to abscisic acid or some stress signal pathways . Previous confirmed that AtbZIP67 played a positive role in ABA-induced AHT1 expression. The up-regulated expression of NtbZIP74 gene in roots may be due to its participation in ABA signaling pathway. In this analysis, NtbZIP76 showed significant responses to heat stress. This gene is homologous to AtbZIP17 (A. thaliana), which plays a vital role in the endoplasmic reticulum heat stress pathway, supporting our findings in tobacco [83,84]. From the expression heat map, we found that most of the NtbZIPs were positively correlated. By considering the interactions between the expression patterns and complex proteins, we could find a feedback mechanism network to coordinate the expression of multiple genes.
In summary, we analysed the whole genome of the bZIP family in tobacco, including gene structure, chromosomal location, sequence homology, synteny, and expression patterns. In this study, a total of 77 NtbZIPs were identified and divided into 11 subfamilies, providing preliminary information for the characterisation of NtbZIP function. Although there were many types of proteins, the exon–intron structure of genes, and the motif arrangement, of bZIP proteins from the same subfamily, supported the classification predicted by the phylogenetic tree. The 77 NtbZIPs were unevenly distributed across 22 tobacco chromosomes. A total of 16 pairs of segmental duplication were identified, illustrating the important role of gene duplication in the expansion of the NtbZIP family. In addition, expression analysis of NtbZIPs in different tissues in response to 6 abiotic stresses implied that NtbZIPs participate in the development of tobacco. NtbZIP49 may be important in the development of flowers and fruits; NtbZIP18 is an important regulator in abiotic stress. These results will be helpful for the further study of the physiological processes performed by the bZIP family in tobacco.
Identification of bZIP family in tobacco
The entire genome sequence of Nicotiana tabacum L. was downloaded from the solgenomics website (https://solgenomics.net/organism/Nicotiana_tabacum/genome). Two BLASTp methods were used to obtain NtbZIPs [85, 86]. Firstly, all AtbZIPs were used to search for possible NtbZIPs from the tobacco genome by using BLASTp (score value ≥ 100 and E value ≤ 1e−10). Secondly, the Hidden Markov Model file of the bZIP domain was downloaded from the Pfam protein family database (http://pfam.xfam.org) . Both HMMER 3.0 (http://hmmer.org/), with a cutoff of 0.01, and SMART (Simple Modular Architecture Research Tool) (https://academic.oup.com/nar/article/49/D1/D458/5940513), were used to identify and verify bZIPs. Furthermore, we analysed the basic characteristics of the identified bZIP proteins using the ExPASY website (https://web.expasy.org/), including the CDS, pI, and Mw.
Exon/intron structures and conserved motif analysis
ClustalW was used to align the default parameters of the multiple amino acid sequences of the bZIPs identified . GeneDoc software (https://genedoc.software.informer.com/) was used to manually adjust the deduced amino acid sequences in the bZIP domain. The exon/intron composition of the bZIPs was analysed by the Gene Structure Display Server (GSDS: http://GSDS.cbi.pku.edu.cn) . MEME (http://meme-suite.org/tools/meme) was used to analyse the motifs of the bZIP proteins [90, 91]. Optimisation parameters were set as follows: repetition times, arbitrary; maximum number of motifs, 10; optimal width of each motif, between 6 and 200 residues [86, 91, 92].
Chromosomal distribution, gene duplication and collinear analysis with other species
All NtbZIPs were mapped to tobacco chromosomes using Circos , according to the physical location information in the tobacco genome database. We used the default parameters of the Multiple Collinearity Scan toolkit (MCScanX) to detect gene duplication . Homology between bZIPs in tobacco and the other 7 species (A. thaliana, O. sativa, F. tataricum, S. lycopersicum, H. vulgare, V. vinifera and S. tuberosum) was analysed through the Dual Synteny Plotter software (https://github.com/CJ-Chen/TBtools).
Phylogenetic analysis and classification of the NtbZIP family
All identified NtbZIPs were divided into several subfamilies, according to the classification of the AtbZIP family. Using the neighbour-joining method of Molecular Evolutionary Genetics Analysis (MEGA) X, the phylogenetic tree was built using Geneious R11 with a BLOSUM62 matrix, using the Jukes Cantor substitution model, a global alignment with free end gaps, and a bootstrap value of 1000. The full-length amino acid sequences of bZIP proteins were compared with the newly identified NtbZIPs for phylogenetic analysis (Additional file 1: Table S1), derived from A. thaliana, O. sativa, F. tataricum, S. lycopersicum, H. vulgare, V. vinifera and S. tuberosum (Uniprot: https://www.uniprot.org/).
Plant materials, growth conditions, and abiotic stress in tobacco
‘K326’, which is a typical, cultivated, common tobacco variety with good yield and quality, was used in this study. ‘K326’ has been grown in the greenhouse of the Guizhou University tobacco research base since 2020. The seeds of ‘K326’ were sown in a special substrate for flue-cured tobacco and raised by floating seedling in a growth room with a 16 h/25 °C day and 8 h/20 °C night regime, and a relative humidity of 75%. We collected the calyx, petal, anther, stigma, leaf, stem, fruit, and root separately from 3 plants showing good and similar growth, 71 days after transplanting, and repeated sampling for three times. To investigate the expression patterns in response to different abiotic stresses, 11 bZIPs were selected for further study. Tobacco plants were subjected to the following abiotic stresses 63 days post-sowing: salt (5% NaCl), water flooding (whole plant), drought (20% PEG6000), UV exposure (70 µW/cm2, 220 V, 30 W), high temperature (38 °C), and low temperature (4 °C). Every abiotic stress treatment was performed with 3 replicates; materials were sampled for qRT-PCR analysis and enzyme activity determination after treatment for 2 h and 24 h. Collected samples were kept at -80 °C until analysis.
Total RNA extraction, cDNA reverse transcription, and qRT-PCR
The RNAprep Pure Plant Kit (TIANGEN, DP432) was used to extract total RNA from samples. The cDNA was generated using the FastKing gDNA Dispelling RT SuperMix kit (TIANGEN, KR118), following manufacturer’s instructions. We selected 11 NtbZIPs that all with close evolutionary relationships to AtbZIPs from different subfamilies to study gene expression patterns. The qRT-PCR primers used to analyse gene expression were designed by Primer Premier 6 (http://www.premierbiosoft.com/) (Additional file 7: Table S5). We used the Actin gene as an internal reference, as it is stably expressed at each growth stage in almost all tissues. The qRT-PCR analysis, performed with Talent qPCR PreMix (SYBR Green) (TIANGEN, FP209), was repeated at least 3 times and the 2−ΔΔCt method was used to caculate the final test results .
DPS (Data Processing System) software was used to analyse the variance of the data, and data were compared with the least significant difference test at the 0.05 and 0.01 levels. OriginPro 2016 software (OriginLab, https://www.originlab.com/) was used for the generation of graphs.
Availability of data and materials
The whole Nicotiana tabacum genome sequence information was obtained from the solgenomics website (https://solgenomics.net/organism/Nicotiana_tabacum/genome) and the website is open to all researchers. The tobacco materials (‘K326’) used in this study were supplied by Prof. Liu Renxiang of Guizhou University. The datasets supporting the conclusions of this article are included in the article and its Additional files.
Basic leucine zipper
Coding sequence length
Data Processing System
Molecular Evolutionary Genetics Analysis
Multiple Expectation maximizations for Motif Elicitation
Protein molecular weight
Real-time quantitative PCR
Simple Modular Architecture Research Tool
Predicted Subcellular location
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This study was financially supported by the National Science Foundation of China (32060510), the Guizhou Province High-level Innovative Talents Training Project Hundred Talents Program (Qiankehepingtairencai5663), the Guizhou Provincial Key Foundation (Qiankehejichu1405), and the Science and Technology Project of Guizhou Tobacco Company (201904).
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Table S1. List of the 77 Nicotianatabacum L. bZIPs identified in this study. bZIP: basic leucine zipper.
Table S2. Analysis and distribution of the conserved motifs in Nicotiana tabacum L bZIP proteins. bZIP: basic leucine zipper.
Table S3. The 16 pairs of segmental duplications in Nicotiana tabacum L. bZIPs.bZIP: basic leucine zipper.
Table S4. One-to-one orthologous gene relationships between Nicotiana tabacum L. and other plants.
Figure S1. The correlation analysis between 8 different tissues and expression patterns of the 11 Nicotiana tabacum L. bZIP genes.
Figure S2. The correlation analysis between 3 different tissues and expression patterns of the 11 Nicotiana tabacum L. bZIP genes under abiotic stresses.
Table S5. Primer sequences used for real-time quantitative PCR.
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Duan, L., Mo, Z., Fan, Y. et al. Genome-wide identification and expression analysis of the bZIP transcription factor family genes in response to abiotic stress in Nicotiana tabacum L.. BMC Genomics 23, 318 (2022). https://doi.org/10.1186/s12864-022-08547-z
- bZIP family
- Nicotiana tabacum L.
- Gene expression patterns
- Abiotic stress