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Article

Genome-Wide Survey and Expression Analysis of the Basic Leucine Zipper (bZIP) Gene Family in Eggplant (Solanum melongena L.)

by
Yan Li
1,2,3,
Guoxin Yao
1,2,
Yafang Tang
1,2,
Xudong Lu
1,2,
Xiu Qiao
2 and
Cheng Wang
1,2,*
1
Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, Hubei Engineering University, Xiaogan 432000, China
2
College of Life Science and Technology, Hubei Engineering University, Xiaogan 432000, China
3
Department of Biology and Chemical Engineering, Weihai Vocational College, Weihai 264200, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(12), 1153; https://doi.org/10.3390/horticulturae8121153
Submission received: 20 October 2022 / Revised: 19 November 2022 / Accepted: 4 December 2022 / Published: 6 December 2022
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Versions Notes

Abstract

:
The transcription factors (TFs) family known as the basic leucine zipper (bZIP) plays a vital role in a variety of biological processes. However, there is no investigation on the bZIP family in the major vegetable crop, eggplant. Here, a total of 71 SmbZIP genes were identified from the eggplant genome and compared with other 18 representative plants. According to the topology of the phylogenetic tree, as well as the categorization and nomenclature of bZIP genes in Arabidopsis and Solanum lycopersicum, the SmbZIP family was classified into 13 groups. Analysis of the chromosome location, motif composition and gene structure of the SmbZIP genes were also performed. Gene duplication analysis revealed that the expansion of the SmbZIP genes was mainly attributed to WGD/segmental duplication. Promoter analysis of the SmbZIP genes and examination of the expression patterns of SmbZIP genes under four abiotic pressures revealed that many SmbZIP genes are related to the control of abiotic stresses. Altogether, the identification, categorization, phylogenetic analysis, chromosome distribution, motif composition, and expression patterns of SmbZIPs were predicted and examined. Importantly, this investigation of SmbZIPs offers a wealth of information that will assist researchers in better understanding their function in eggplant.

1. Introduction

Transcription factors (TFs) attach to target genes to activate or inactivate their expression in response to various biological processes and stress responses [1]. Among all TFs, the basic leucine zipper (bZIP) family is one of the largest and most diverse families [2]. The bZIP TFs are named based on the highly conserved bZIP domain which is 60–80 amino acids in length. Two structural components make up the bZIP domain: a basic region and a leucine zipper motif [2,3]. The basic region of about 16 amino acids is highly conserved and residues with an invariant N-x7-R/K-x9 motif for nuclear localization and sequence-specific DNA binding [4,5]. The leucine zipper motif is less conserved, containing heptad repeats of leucine (Leu) or other bulky hydrophobic amino acids which is involved in specific recognition and homo- and/or heterodimerization of bZIP proteins [6]. Plant bZIP proteins primarily bind to the A-box (TACGTA), C-box (GACGTC), and G-box (CACGTG), which are DNA sequence motifs with an ACGT core [7]. The basic region’s N-terminal half inserts into the main groove of double-stranded DNA after DNA binding, while the Leu zipper’s C-terminal half mediates dimerization to generate a superimposed coiled-coil shape [6,8].
Due to the completion of genome sequencing in many species, members of the bZIP TF family have been discovered and predicted in numerous plants. For example, 247 bZIP genes have been reported in rapeseed (Brassica napus L.) [9], whereas, 160 in soybean (Glycine max (Linn.) Merrill) [10], 86 in Poplar (Populus trichocarpa (Torr. & Gray)) [11], 227 in wheat (Triticum aestivum L.) [12], 89 in rice (Oryza sativa L.) [13], 78 in Arabidopsis (Arabidopsis thaliana (L.) Heynh) [14], and 55 in grape (Vitis vinifera L.) [15] have been reported. In general, based on sequence similarity in their fundamental regions and conserved motifs, putative bZIP genes have been classified into tens of categories. Based on DNA-binding specificity and amino acid sequence, the predicted bZIP proteins of O. sativa were divided into 11 groups [13], while the AtbZIP family was categorized into 13 groups in A. thaliana [14]. Although there are fewer bZIP family groups in O. sativa than in A. thaliana, the association among these groups is comparable. In addition, interspecies clustering revealed that homologous bZIPs from both species belonged to the same group [16].
The bZIP family genes are crucial for plant developmental and physiological processes. For example, rice OsbZIP48 could regulate the zinc deficiency response [17]. Overexpression of ZmbZIP4 in maize resulted in a better root system with more lateral roots and longer primary roots [18]. The bZIP proteins also mediate plant responses to abiotic stresses, such as drought and salt stresses. For instance, overexpression of GmbZIP2 in Arabidopsis and soybean significantly improved plant resistance to drought and salt stresses [19]. Apple C/S1 bZIP network could control MdIPT5b to alter apple species’ susceptibility to drought [20]. In wheat, enhancing TabZIP15 expression resulted in increased resistance to salt stress [21]. In Arabidopsis, AtbZIP36, 37, and 38, genes were involved in response to the ABA signaling, dehydration, and salinity [22]. Additionally, the bZIP transcription factor TGA5 (AtbZIP26), which interacted highly with NIM1/NPR1, conferred SAR-independent resistance to Peronospora parasitica [23]. Similarly, in rice, OsABF2 regulated the expression of genes that respond to abiotic stresses through an ABA-dependent pathway [24]. OsbZIP23 has been reported to be a central regulator in ABA signaling and biosynthesis, and overexpression of OsbZIP23 displayed noticeably enhanced drought and salt tolerance [25]. Together, these pieces of evidence suggest that bZIP genes play a significant role in both development and biotic/abiotic stresses.
Eggplant (Solanum melongena L.), as an agronomically important solanaceous crop, is cultivated and consumed worldwide. Eggplants are utilized not only as a food source but also as a source of chlorogenic acid, which is good for human health [26]. Despite the economic and nutritional importance of eggplant, as well as the crucial role of bZIP TFs in plant growth and stress responses, no evidence of bZIP family genes being identified and characterized in eggplant has been reported. The eggplant whole-genome sequence [27] provides excellent resources for learning more about gene function and evolution. In this current study, we investigated the phylogenetic relationships, chromosome location, gene structure, motif composition, cis-elements distribution, gene duplications, and interaction network of the bZIP gene family in eggplant. Moreover, 25 SmbZIP genes were chosen for an evaluation of their expressional profile under four various abiotic stresses. These findings will aid future study into the SmbZIP gene in functional genomic investigations, as well as their application in this vital crop under abiotic stress.

2. Materials and Methods

2.1. Investigation of bZIP Genes Family in Eggplant and Other Plant Species

The genome sequences of eggplant were downloaded from Eggplant Genome Database [27]. The hidden Markov Model profile of bZIP (PF00170, PF03131, PF07716) domain obtained from Pfam database [28] was used as queries to search predicted SmbZIPs from eggplant genome using the HMMER software (version: 3.0) [29]. Subsequently, the Arabidopsis bZIPs [14] were used to identify the bZIP in eggplant using the BLASTP search method with a threshold of 1 × 10−10. Finally, SMART [30] and Pfam databases [28] were used to confirm these sequences.
The bZIP proteins from the other 18 typical plants were acquired in a few different ways. The bZIP proteins from Ceratopteris richardii were obtained using the same procedure as described above. The bZIP proteins of Carica papaya and Theobroma cacao were obtained from PlantRegMap [31]. The bZIP proteins of following species were retrieved from previous analyses: Arabidopsis thaliana [14], Malus domestica [32], Cucumis sativus [33], Medicago truncatula [34], Populus trichocarpa [11], Vitis vinifera [15], Selaginella moellendorffii [35], Solanum lycopersicum [36], Sorghum bicolor [37], Ginkgo biloba [38], Chlamydomonas reinhardtii [35], Physcomitrella patens [35], and Cyanidioschyzon merolae [35]. The evolutionary links between these species were established using the PGDD database [39] and earlier studies [40,41].

2.2. Phylogenetic Characterization of bZIP Gene Family

Five species: eggplant, A. thaliana (a rosid eudicot), S. lycopersicum (a related asteroid eudicot), Aquigelia coerulea (a basal eudicot), and Amborella trichopoda (a basal angiosperm), were selected to constructed phylogenetic tree. The AtbZIP and SlbZIP proteins from A. thaliana and S. lycopersicum were retrieved from previous studies [14,36], while the bZIP protein from A. coerulea, and A. trichopoda were acquired using the same procedure as described above.
T-Coffee [42] was used to align the bZIP protein sequences from these five species. The alignment sequences were then manually modified using the Jalview program [43]. The most accurate protein evolution model was selected using the ModelFinder [44]. The phylogenetic tree was constructed using IQ-TREE [45] with a bootstrap value of 5000. Finally, the phylogenetic tree was visualized and embellished with iTOL (https://itol.embl.de/ (accessed on 12 November 2022)).

2.3. Phylogenetic, Motif Composition, Gene Structure, and Physicochemical Analysis of SmbZIP Proteins

Gene structure analysis was performed using TBtools software (version: 1.1047) [46] after downloading a generic file format (GFF) from the eggplant genome database [27]. The MEME suite was used to determine the motif composition of bZIP proteins [41], with following setting: the number of motifs was set to 20 and the width was ranged from 40–100, while the other parameter was set to default. Finally, an XML file was retrieved from the MEME server. With the use of the TBtools software, the GFF and XML files were used to visualize gene structure and motif composition. In addition, the ProtParam server was used to determine the molecular weight (MW), isoelectronic points (PIs), and grand average of hydropathicity index (GRAVY) of each bZIP protein in eggplant. The WOLF PSORT was used to undertake subcellular prediction analysis [47].

2.4. Chromosome Distribution, Gene Duplication, and Synteny Analysis of SmbZIPs in Eggplant

All SmbZIP genes were mapped on the twelve eggplant chromosomes based on their position annotation, the locations of the SmbZIP genes were visualized using the TBtools software [46]. BLASTp was used to look for putative homologous gene pairs (E < 1 × 10−5) across the whole eggplant genome. The Multiple Collinearity Scan Toolkit (MCScanX) was run with the default settings on the probable homologous gene pairs in order to find syntenic chains [48]. MCScanX was utilized to further differentiate between whole-genome duplication (WGD)/segmental, scattered, proximal, and tandem duplication events in the SmbZIP gene family. Using the PAML tool, Ka and Ks values were calculated for gene pairs found in the same synteny block [49].

2.5. Promoter Analysis of SmbZIP Genes

We explored all the SmbZIP promoter sequences (2000 bp upstream of the transcription start site). PlantCARE was used to analyze the promoter sequence to identify each gene’s cis-regulatory elements [50]. With the use of the TBtools software, the GFF and PlantCARE output files were used to visualize cis-regulatory elements.

2.6. Prediction of the SmbZIPs Protein-Protein Interaction Network

To forecast protein interactions, protein-protein interaction (PPI) investigations of the SmbZIP family were carried out on the STRING website [51] (version: 11.5). S. lycopersicum was employed as the model and the SmbZIP amino acid sequences as the query sequences. The online program was run with the following settings: the ‘required score’ box had the maximum confidence level set to 0.9, while all other boxes had default values.

2.7. Plant Materials, Growth Conditions, and Stress Treatments

The plant material used in this research was eggplant cultivar ‘bailong’. Plants were grown in plastic pots with a soil–vermiculite (3:1) mixture. The growth conditions were set at 25/20 °C with a photoperiod of 16/8 h for day/night. Various abiotic stress treatments were carried out with five-leafed plants. The plants were irrigated with 15% (w/v) polyethylene glycol (PEG) 6000 and 250 mM NaCl for drought and NaCl treatment, respectively. For temperature stress, whole plants were exposed to 4 °C or 42 °C. Leaves from control and stress-treated plants were harvested 0, 1, 6, and 12 h after application of the stress treatments. Each analysis included triplicate samples and samples from five different plants were regarded as a biological replicate. All samples were immediately frozen in liquid nitrogen, and then stored at −80 °C for RNA isolation.

2.8. RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNA was extracted by RNA Total kit (TaKaRa, Dalian, China), according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA by using a Prime Script RT reagent kit (TaKaRa, Dalian, China). The specific primers for qRT-PCR were designed by Beacon Designer 8 (Table S1). The SmEF1a (Smechr1201845.1) was used as an internal control. Real-time qPCR was performed on the StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) with the following cycling profile: 95 °C for 30 s, 95 °C for 5 s (40 cycles), and 60 °C for 30 s. Three biological and three technical replicates were used in each RT-qPCR experiment. The reaction mixture contained 10 µL of TB Green® Premix Ex TaqTM II (2×) (TaKaRa, Dalian, China), 0.8 µL of each primer (10 µM), 2 µL of diluted cDNA (1:10), 0.4 µL of ROX Reference Dye (50×), and 6 µL of dH2O in a final volume of 20 µL. The results were calculated using the 2−ΔΔCt method [52].

3. Results

3.1. Identifcation and Chromosome Distribution of the SmbZIP Gene Family in Eggplant

There are 71 SmbZIP family proteins identified in eggplant (Table S2). The bZIP genes in eggplant were assigned unique names and renamed as SmbZIP1-SmbZIP71 based on their chromosomal positions from Chr1-12. Protein length (aa), molecular weight (MW, kDa), isoelectric point (PIs), grand average of hydropathicity (GRAVY), and subcellular prediction were all examined for these genes (Table S2). The SmbZIP proteins have an amino acid residue count that varied from 125 (SmbZIP10) to 1835 (SmbZIP57). The MW of SmbZIP proteins varied from 13.04 kDa (SmbZIP10) to 204.20 kDa (SmbZIP57). The putative PIs of the 71 family members ranged from 5.13 (SmbZIP38) to 9.71 (SmbZIP7). All SmbZIP proteins had a hydropathicity (GRAVY) value less than zero, indicating that they are all hydrophilic (Table S2). The majority of SmbZIP genes were found in the nucleus, cytoplasm, chloroplast, plasma membrane, and vacuole, according to the subcellular localization analyses.
As shown in Figure 1, all 12 chromosomes had SmbZIP genes, although their congregate area and amount were unequal. The number of genes found on each chromosome varies, with the maximum 13 genes found on chromosome 1, 8 each on chromosome 4 and 6, and only 1 gene found on chromosome 3. SmbZIP gene content per chromosome ranged from 0.07% on chromosome 5 to 0.36% on chromosome 12 (Table S3).

3.2. Phylogenetic Analysis of SmbZIP Gene Family and Comparative Analyses

Forty-nine and thirty-nine bZIP proteins were identified in A. coerulea and A. trichopoda (Table S4). According to the topology of the phylogenetic tree, as well as the categorization and nomenclature of bZIP genes in A. thaliana and S. lycopersicum, 306 members of the bZIP family from five diverse species were divided into 13 groups (Figure 2, Table 1). All groups in our study were supported by previous study in A. thaliana (A-K, M, S), and the roman numerals in parentheses shown in Figure 2 expressed the previous grouping in S. lycopersicum (I–XI). Eggplant, A. thaliana, and A. coerulea had all 13 groups, while A. trichopoda lacked Group B and Group M, and S. lycopersicum lacked Group K. Moreover, the results show that SmbZIP genes have an uneven distribution. Group S, for example containing 15 SmbZIP genes, is the largest group, whereas Group A, B, and E had 12, 3, and 6 SmbZIP genes, respectively (Figure 2, Table 1).
Moreover, a comparative genomic analysis was also carried among bZIP in 19 representative plant species (Figure 3). Our research revealed that bZIP gene numbers in algae, bryophytes, and lycophytes are less than those in angiosperms, which have undergone numerous whole genome duplication events (Figure 3). The number of bZIP genes in single-celled C. reinhardtii and fern S. moellendorffii were 7 and 8, respectively. However, the genome of the moss P. patens encodes up to 40 bZIP members, indicating a considerable expansion after this species split from green algae. Possibly due to the triplication of the Solanaceae genome, the genome sizes of S. lycopersicum (760.0 MB) and S. melongena (1073.1 MB) were larger than most angiosperm species (excluding M. domestica). However, their genome has a lower density of bZIP genes than all angiosperm plants, implying that many SmbZIPs were lost during polyploidy speciation. Additionally, G. biloba and C. richardii have the lowest densities of bZIP genes compared to all other species because of their enormous genome sizes.

3.3. Gene Structure and Protein Motif Analysis of bZIP Gene Family in Eggplant

To better analyze the conservation and diversification of bZIP genes in eggplant, the gene structure and conserved motifs were conducted (Figure 4 and Figure S1). The maximum-likelihood phylogenetic tree was constructed using the SmbZIP domain sequences (Figure 4a). Gene structure analysis revealed that 71 SmbZIP genes possessed exons varying from 1 to 15 (Figure 4c). Sixteen SmbZIP genes, including SmbZIP3, 7, 10, 11, 16, 17, 20, 21, 25, 27, 32, 51, 61, 63, 64, and 66, contained only one exon and no intron. As anticipated, gene structure analysis revealed that the majority of individuals belonging to the same group had similar intron/exon compositions, including the quantity and length of exons. For instance, all members of Group S have fewer than 2 exons, but all members of Group G have more than 10 exons. The MEME analysis showed that different groups showed different motif profiles, and the components of the conserved motifs for proteins in the same group were similar (Figure 4b). All of the SmbZIPs contained motif 1 or 9, which were annotated by the tool SMART as bZIP domain. Moreover, an examination at motif distribution revealed that some motifs were exclusive to certain groups of the SmbZIP family. For instance, motifs 2 and 3 were unique to Group D, and motif 4 and 16 were only present in Group G. Additionally, motif 15 was unique to Group C, while motif 18 was only seen in Group E. Thus, our findings further support the phylogenetic relationship of these SmbZIP genes by showing that SmbZIP genes in the same group shared a comparable motif composition as well as similar exon and intron distribution.

3.4. Gene Collinearity, Duplication, and Ka/Ks Analysis of SmbZIPs

Gene duplication events including WGD/segmental duplication, proximal duplication, and dispersed duplication all aid in accelerating the evolution of gene families. In the current work, we investigated the origins of duplicate genes for the SmbZIP gene family. Members of the SmbZIP gene family were categorized into one of the following groups: tandem, proximal, dispersed, WGD/segmental duplication, and singleton. Intriguingly, 40 (56.3%), 25 (35.2%), and 6 (8.5%) of the SmbZIPs in eggplant were duplicated and retained through WGD/segmental duplication, dispersed, and tandem, respectively (Table S2, Figure S3). In contrast, none of the SmbZIP genes in our results were derived from proximal or singletons.
Furthermore, we studied the collinearity relationships among SmbZIP genes (Figure 5). Among SmbZIPs, high conservation was detected and a total of 28 pairs were detected as collinear (Figure 5 and Table S5). Subsequently, these gene pairs were analyzed for synonymous (Ks) and non-synonymous (Ka) mutations (Table S5). Purifying selection (Ka/Ks < 1), positive selection (Ka/Ks > 1), and neutral selection (Ka/Ks = 1) are common types of selection pressure associated with genes during evolution. All 24 SmbZIP gene pairs in the current investigation had Ka/Ks ratios lower than 1, which indicates that purifying selection and reduced divergence occurred among them. Collinearity analysis was also used to investigate the orthologous relationships of the bZIP family genes in rice, Arabidopsis, and eggplant (Figure S4, Tables S6 and S7). There were 36 orthologous bZIP gene pairs between eggplant and Arabidopsis and 20 orthologs between eggplant and rice.

3.5. Promoter Analysis of SmbZIP Family Genes

Cis-regulatory elements control the transcription process; the examination of promoter cis-acting elements is a valuable research tool to investigate the gene regulation mechanisms. In the present study, we identified the cis-acting elements in the 2000 bp upstream of the transcription start site of SmbZIP genes (Figure S2, Table S8). The results revealed that most cis-acting elements were mainly related to three different categories: hormone-responsive elements (CGTCA-motif, AuxRR-core, GARE-motif, P-box, SARE, ABRE, TCA-element, TGACG-motif, TGA-element); stress-responsive elements (LTR, MBS, TC-rich repeats, ABRE); and light-responsive elements (3-AF1 binding site, AAAC-motif, ACE, G-box, GT1-motif, MRE, Sp1, TATC-box). It is well recognized that SmbZIP genes play a critical role in regulating responses to abiotic stresses. Thus, we focused on SmbZIP genes, which contained stress-responsive elements (ABRE, MBS, LTR, TC-rich repeats). Previous research has demonstrated the importance of the ABRE motif for ABA-dependent drought defense, the MBS motif for the response to drought, LTR motif for the response to cold, and the TC-rich repeats for the regulation of the gene expression of plant defense and stress responsiveness. A total of 66 SmbZIPs contained stress-responsive elements, of which 25 SmbZIP contained at least four stress-responsive elements (Figure S5). Considering the distribution of cis-elements in the promoter of these genes, we speculate that these SmbZIPs may play a significant role in controlling the expression of the related genes for abiotic stresses.

3.6. Interaction Network of bZIP Proteins in Eggplant

For the purpose of researching the family’s regulatory pathways, it is crucial to comprehend the functional interactions between the SmbZIP proteins. Thus, we construct an SmbZIP protein interaction network based on the orthologouses in Solanum lycopersicum, systematically analyzing the interactions of SmbZIP proteins. The orthologous proteins with the highest bit score were considered STRING proteins, and 33 SmbZIP proteins were chosen because of the consideration of reliability (Figure 6, Table S9). Large protein-protein interaction pairings were predicted in SmbZIPs, as illustrated in Figure 6, suggesting that SmbZIP family members might dimerize to influence gene expression. Additionally, a number of SmbZIPs were shown to be key nodes in the whole network, suggesting that they may be engaged in a variety of physiological processes. SmbZIP45, for instance, was speculated to be related to SmbZIP9, 15, 18, 22, 23, 48, 56, and 59. On the other hand, some SmbZIPs, such as SmbZIP40-SmbZIP53, SmbZIP36-SmbZIP22, and SmbZIP60-SmbZIP10 pairs, could only communicate with particular members. These findings therefore show how SmbZIP protein family members may form functional transcription factor complexes, regulating the expression of numerous genes in eggplant.

3.7. Expression Pattern of SmbZIPs under Stresses

It is well recognized that the bZIP gene family has significant regulatory functions in regulating responses to various abiotic stresses. To identify how SmbZIPs are involved in various stress responses, 25 SmbZIPs containing at least four stress-responsive elements were selected as candidate genes (Figure S5), and their expression profiles under four stress treatments (salt, drought, heat, and cold) were examined using qRT-PCR in the eggplant varieties ‘bailong’. The findings demonstrated that 25 SmbZIP genes reacted differentially to the treatments (Figure 7, Table S10). The expression levels of SmbZIP14, 44, and 57 were significantly upgraded by the cold treatment (Figure 7a). Under heat treatment, most SmbZIP genes were either down-regulated or did not change appreciably, while SmbZIP8 and 25 were obviously up-regulated during the continuous time course (Figure 7b). Under NaCl stress, the expression of SmbZIP1, 14, 47, and 71 were up-regulated at 1, 6, and 12 h, while SmbZIP19 and 60 showed peak expression at the 6 h and 12 h time point, respectively (Figure 7c). Under PEG stress, the expression levels of SmbZIP2 and 59 were up-regulated at 1, 6, and 12 h, while SmbZIP14 and 47 were only obviously up-regulated at 1 and 12 h (Figure 7d). Moreover, the expression of SmbZIP6, 15, 25, 28, 38, 40, 54, and 61 were down-regulated or scarcely changed under all treatments, while SmbZIP14, according to the above result, responded to all treatments other than heat.

4. Discussion

The bZIP family, one of the most essential TFs families, has been linked to a variety of important functions, including plant development, organ differentiation, and stress response [14,53]. In the present study, we analyzed the bZIP gene family in eggplant and in 18 other species, including 16 higher plants and 2 lower plants, a total of 1235 bZIP genes are identified and analyzed. Since the number of bZIP genes in C. reinhardtii and S. moellendorffii were far less than that in P. patens, we inferred that the bZIP genes in P. patens may experience a considerable increase following the divergence from green algae (Figure 2). P. patens has a position on the evolutionary tree that allows reconstruction of evolutionary changes in genomes associated with the conquest of land using comparisons with aquatic algae and vascular plants [54]. The expansion of the bZIP family in P. patens demonstrates the importance of bZIP proteins in various physiological activities, notably in complex function. In addition, the bZIP proteins in G. biloba and C. richardii were fewer than the majority of species while having a genome that was 20–80 times larger, indicating that the number of bZIP genes was not correlated with genome size. The density of bZIP proteins in Brassicales (0.578 for Arabidopsis thaliana, 0.479 for Brassica rapa) was higher than in other species used in this analysis, suggesting that bZIP proteins may play a key role in Brassicales species.
A total of 71 SmbZIP genes were identified in this study. These genes were then classified into 13 groups according to the topology of the phylogenetic tree, which was supported by a previous study that classified AtbZIPs into 13 subfamilies [14], while the grouping of SlbZIPs in S. lycopersicum differed slightly from the prior work [36]. In order to make comparisons easier, we labeled the previously identified clades in the trees shown in Figure 2. The differences were as follows: the former Group IV (Four) was further divided into three groups (Groups C, M, and S); the former Group V (Five) and Group X (Ten) were combined as a new group (Group E). Due to the grouping disparities with S. lycopersicum, we used gene structure and protein motif analysis to support our group designations (Figure 4). The results revealed that the exon and intron distributions and motif compositions of the majority of individuals of a particular group were comparable. This significant agreement with evolutionary relationships proved that our categorization was accurate.
In angiosperm genomes, there are five major types of gene duplication that each contribute to the expansion of plant protein-coding gene families: WGD/segmental, tandem, proximal, dispersed duplication, and singleton [55,56,57]. Meanwhile, the WGD/segmental and tandem duplication events are the most important contributors to gene family expansion during evolution [48]. Some transcription factor gene families, including GRAS, bHLH, and WRKY, most likely grew by WGD/segmental duplication [58,59,60,61,62], while the expansion of some other transcription factor gene families, such as the NBS-LRR gene family and the ERF gene family, is a result of tandem duplication [63,64,65]. Our research indicated that WGD/segmental duplication had a crucial role in the growth of the SmbZIP family genes in eggplant, since 56.3% of SmbZIP genes were duplicated and maintained from this process. This result is in agreement with earlier research on poplar [11], tobacco [66], walnut [67], and pear [68]. Subfunctionalization and neofunctionalization occur concurrently with gene expansion. During selection and evolution, duplicated genes can be stably maintained if they differ in parts of their functions, which might explain why the segmental duplicated gene pair SmbZIP19/SmbZIP25 from group S responds to abiotic stressors with variable levels of expression. In view of the importance of gene duplication, we also assessed the rate of synonymous (Ks) to non-synonymous (Ka) mutation among the WGD/segmental and tandem gene duplications. It is widely known that genes are often connected with three varied types of selection, including purifying selection (Ka/Ks < 1), positive selection (Ka/Ks > 1), and neutral selection (Ka/Ks = 1) [69]. In our research, The Ka/Ks ratio of all WGD/segmental and tandem duplication gene pairs were less than 1.00, implying that they were subjected to strong Darwinian purifying positive selection.
The principal mechanism for controlling the expression of genes in plants is transcriptional regulation. In eukaryotes, the interaction between TFs and promoter binding sites is crucial for regulation at the transcriptional level. External signals induce and activate inducible promoters, and the cis-acting parts of inducible signals in promoters are specific and consistent. For example, several important cis-elements were identified, including stress-responsive elements (LTR, MBS, TC-rich repeats, ABRE), hormone-responsive elements (AuxRR-core, CGTCA-motif, GARE-motif, P-box, SARE, ABRE, TCA-element, TGACG-motif, TGA-element), etc. The cis-acting element in our study revealed that SmbZIP genes are regulated by a variety of hormones and are linked to a variety of stresses and that they may be advantageous in boosting eggplant stress resistance. These stress-related cis-acting elements enable plants to react quickly and control the expression of associated genes, increasing their environmental resilience. To evaluate how SmbZIP genes react to various stresses, 25 SmbZIPs that had at least four stress-responsive elements were examined for their expression patterns. For each treatment, more than one gene showed significant expression during the continuous time course. For instance, SmbZIP8 and 25 were obviously up-regulated under heat treatment during the entire time course, while the expression of SmbZIP1, 47, and 71 were up-regulated at 1, 6, and 12 h under NaCl stress. Two AtbZIP genes, AtbZIP24 and 62 in Arabidopsis, were discovered to be up-regulated in response to salt stress [53]. In this study, SmbZIP1 and 47, which are most closely linked to AtbZIP24 and 62, were upregulated when exposed to NaCl treatment (Figure 4a). In particular, SmbZIP14 was expressed at relatively high levels under cold, NaCl, and PEG treatments, which indicated that it might be crucial for eggplant’s reactions to abiotic stress. These findings add to the growing body of evidence that bZIP genes are critical in the face of a variety of environmental stressors.

5. Conclusions

In this research, 71 bZIP genes were identified in the eggplant genome and were categorized into 13 groups. Evolutionary relationships, motif composition, and gene structure of SmbZIP genes were also identified and investigated. Additionally, the expansion of the bZIP genes in eggplant was attributed to WGD/segmental duplication rather than other duplication types, and selective pressure analysis (Ka/Ks) implying all segmental duplications SmbZIPs pairs suffered purifying selection. Promoter analysis of the SmbZIPs, together with the expression patterns of SmbZIPs under four abiotic stresses, provided a basic resource for the investigation of the molecular control of eggplant stress resistance. Our research represents the first systematic and thorough examination of bZIP genes in eggplant, while more research on the functional mechanisms of SmbZIPs is required. Nonetheless, this research provides a preliminary exploration of SmbZIPs and lays the groundwork for future research into bZIP genes in other species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8121153/s1, Table S1: The primer sequences used for quantitative real-time PCR amplification of actin and 25 SmbZIP genes; Table S2: The basic information of bZIP genes identified in eggplant; Table S3: The distribution ratio of SmbZIP genes on each chromosome in eggplant; Table S4: The bZIP genes in Aquigelia coerulea and Amborella trichopoda; Table S5: Segmental and tandem duplications gene pairs; Table S6: The orthologous relationships of the bZIP genes between Arabidopsis and eggplant; Table S7: The orthologous relationships of the bZIP genes between rice and eggplant; Table S8: Cis-elements predictions of bZIP in eggplant; Table S9: The information of 33 SmbZIP proteins and their corresponding STRING protein in Solanum lycopersicum; Table S10: Relative expression of SmbZIP genes under different abiotic stresses, as determined by qRT-PCR; Figure S1: Sequence logos of SmbZIP domains; Figure S2: Cis-element analysis of promoter (2000 bp upstream) in SmbZIP genes; Figure S3: Proportion of genes originating from different replication events; Figure S4: The orthologous relationships of bZIP genes among eggplant, Arabidopsis, and rice. (a) The orthologous relationships of bZIP genes between eggplant and Arabidopsis; (b) The orthologous relationships of bZIP genes between eggplant and rice. The black lines indicated the orthologous bZIP gene pairs. A color box with a number inside it represents each chromosome. Sm: S. melongena; At: Arabidopsis; Os: Rice; Figure S5: The SmbZIPs that containing at least 4 stress-responsive elements.

Author Contributions

Conceptualization, C.W. and Y.L.; methodology, C.W.; software, G.Y.; validation, C.W., Y.L., and X.Q.; formal analysis, Y.T.; investigation, X.L.; resources, C.W.; data curation, Y.L.; writing—original draft preparation, Y.L.; writing—review and editing, C.W.; visualization, G.Y.; supervision, X.L.; project administration, Y.T.; funding acquisition, C.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Xiaogan Natural Science Project, grant number XGKJ2021010101; the Provincial Teaching Research Project of Hubei Universities, grant number 2020634.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Distribution of SmbZIP genes on eggplant chromosomes. The relative length of a chromosome serves as a measure of its size. Red bars indicate tandemly duplicated genes.
Figure 1. Distribution of SmbZIP genes on eggplant chromosomes. The relative length of a chromosome serves as a measure of its size. Red bars indicate tandemly duplicated genes.
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Figure 2. Phylogenetic analysis of bZIP proteins in eggplant, A. thaliana, S. lycopersicum, A. coerulea, and A. trichopoda. The phylogenetic tree was constructed using IQ-TREE with a bootstrap value of 5000. The phylogenetic tree was constructed based on the 306 bZIP domain sequences. The letters or roman numbers that follow the names of the S. lycopersicum or Arabidopsis bZIP genes denote categorization in previous studies.
Figure 2. Phylogenetic analysis of bZIP proteins in eggplant, A. thaliana, S. lycopersicum, A. coerulea, and A. trichopoda. The phylogenetic tree was constructed using IQ-TREE with a bootstrap value of 5000. The phylogenetic tree was constructed based on the 306 bZIP domain sequences. The letters or roman numbers that follow the names of the S. lycopersicum or Arabidopsis bZIP genes denote categorization in previous studies.
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Figure 3. Summary of bZIP genes family among 19 genomes of different plant species.
Figure 3. Summary of bZIP genes family among 19 genomes of different plant species.
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Figure 4. Phylogenetic relationship, conserved motif compositions and gene structure of SmbZIP proteins. (a) The maximum-likelihood tree based on the 71 SmbZIP domain sequences with 5000 bootstrap replicates. (b) The conserved motifs in the SmbZIP proteins were detected by MEME analysis and represented by color boxes. (c) The gene structure of SmbZIPs were analyzed by TBtools. Introns and exons are represented by black lines and orange round-cornered rectangles, respectively.
Figure 4. Phylogenetic relationship, conserved motif compositions and gene structure of SmbZIP proteins. (a) The maximum-likelihood tree based on the 71 SmbZIP domain sequences with 5000 bootstrap replicates. (b) The conserved motifs in the SmbZIP proteins were detected by MEME analysis and represented by color boxes. (c) The gene structure of SmbZIPs were analyzed by TBtools. Introns and exons are represented by black lines and orange round-cornered rectangles, respectively.
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Figure 5. Genomic locations of SmbZIP genes and segmentally duplicated gene pairs in the eggplant genome (a) and Ka, Ks, and Ka/Ks ratio of segmental duplicate genes (b). The 24 segmentally duplicated SmbZIP gene pairs are indicated by the black lines. A color box with a number inside it represents each chromosome. The box plots are exhibiting the distributions of Ka, Ks, and Ka/Ks values among paralogs. The small square and the line in the box represent average and median values of the Ka, Ks, and Ka/Ks values, respectively. Sm: Solanum melongena; At: Arabidopsis thaliana; Os: Oryza sativa.
Figure 5. Genomic locations of SmbZIP genes and segmentally duplicated gene pairs in the eggplant genome (a) and Ka, Ks, and Ka/Ks ratio of segmental duplicate genes (b). The 24 segmentally duplicated SmbZIP gene pairs are indicated by the black lines. A color box with a number inside it represents each chromosome. The box plots are exhibiting the distributions of Ka, Ks, and Ka/Ks values among paralogs. The small square and the line in the box represent average and median values of the Ka, Ks, and Ka/Ks values, respectively. Sm: Solanum melongena; At: Arabidopsis thaliana; Os: Oryza sativa.
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Figure 6. Functional interaction network of SmbZIP proteins in eggplant according to orthologues in S. lycopersicum.
Figure 6. Functional interaction network of SmbZIP proteins in eggplant according to orthologues in S. lycopersicum.
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Figure 7. Expression analysis of 25 selected SmbZIP genes under four abiotic stresses. (a) The relative expression ratio of 25 selected SmbZIP genes under cold stress. (b) The relative expression ratio of 25 selected SmbZIP genes under heat stress. (c) The relative expression ratio of 25 selected SmbZIP genes under NaCl stress. (d) The relative expression ratio of 25 selected SmbZIP genes under PEG stress. Each treatment contains three time points: 1 h, 6 h, and 12 h.
Figure 7. Expression analysis of 25 selected SmbZIP genes under four abiotic stresses. (a) The relative expression ratio of 25 selected SmbZIP genes under cold stress. (b) The relative expression ratio of 25 selected SmbZIP genes under heat stress. (c) The relative expression ratio of 25 selected SmbZIP genes under NaCl stress. (d) The relative expression ratio of 25 selected SmbZIP genes under PEG stress. Each treatment contains three time points: 1 h, 6 h, and 12 h.
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Table
Table 1. The categorization of bZIP genes in five species.
Table 1. The categorization of bZIP genes in five species.
SpeciesNumber of Genes Included
A (VI)B (III)C (IV)D (VII)E (V + X)F (VIII)G (I)H (II)I (IX)J (XI)KM (IV)S (IV)Total
Eggplant123312625172121571
Arabidopsis thaliana1334106352121111778
Solanum lycopersicum123312516172041369
Aquigelia coerulea7128315261111149
Amborella trichopoda802552424120439
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Li, Y.; Yao, G.; Tang, Y.; Lu, X.; Qiao, X.; Wang, C. Genome-Wide Survey and Expression Analysis of the Basic Leucine Zipper (bZIP) Gene Family in Eggplant (Solanum melongena L.). Horticulturae 2022, 8, 1153. https://doi.org/10.3390/horticulturae8121153

AMA Style

Li Y, Yao G, Tang Y, Lu X, Qiao X, Wang C. Genome-Wide Survey and Expression Analysis of the Basic Leucine Zipper (bZIP) Gene Family in Eggplant (Solanum melongena L.). Horticulturae. 2022; 8(12):1153. https://doi.org/10.3390/horticulturae8121153

Chicago/Turabian Style

Li, Yan, Guoxin Yao, Yafang Tang, Xudong Lu, Xiu Qiao, and Cheng Wang. 2022. "Genome-Wide Survey and Expression Analysis of the Basic Leucine Zipper (bZIP) Gene Family in Eggplant (Solanum melongena L.)" Horticulturae 8, no. 12: 1153. https://doi.org/10.3390/horticulturae8121153

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