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Article

Identification of the bZIP Gene Family and Investigation of Their Response to Drought Stress in Dendrobium catenatum

by
Peng Wang
1,†,
Yuxin Li
1,†,
Tingting Zhang
1,2,
Yuqian Kang
1,
Wei Li
3,
Jian Wang
1,
Wengang Yu
1,* and
Yang Zhou
1,*
1
Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, School of Horticulture, Hainan University, Haikou 570228, China
2
Xiangyang Academy of Agricultural Sciences, Xiangyang 441057, China
3
State Key Laboratory of Cotton Biology, Institute of Cotton Research of the Chinese Academy of Agricultural Sciences, Anyang 455000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(1), 236; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13010236
Submission received: 9 December 2022 / Revised: 9 January 2023 / Accepted: 10 January 2023 / Published: 12 January 2023
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Dendrobium catenatum is a valuable Chinese herbal medicine that naturally grows on cliffs and tree trunks and is often threatened by adverse environmental conditions. The bZIP transcription factor is known to play a critical role in the response of plant to stress. However, the functions of the bZIP gene family in D. catenatum are poorly understood. In this study, 62 bZIP genes were identified from D. catenatum, which encoded proteins with an amino acid number of 130~692, a molecular weight of 15.24 to 74.94 kDa, and an isoelectric point of 5.13 to 11.58. The bZIP family can be divided into 10 subgroups by evolutionary tree analysis, and the conserved motifs of each protein subgroup were similar. The exon number of bZIP genes ranged from 1 to 12 as shown by gene structure analysis. DcbZIP promoter prediction analysis identified 21 cis-acting elements. The expression of DcbZIP genes under drought treatment was analyzed using the public RNA-seq data, and 33 upregulated genes were further screened. A co-expression network analysis revealed that 17 core genes were closely correlated with other genes and their expression was measured using RT-qPCR. The results showed that DcbZIP6, DcbZIP34, DcbZIP42 and DcbZIP47 are the main contributors to drought tolerance in D. catenatum. In summary, we identified candidate bZIP genes in D. catenatum with a apotential contribution to drought stress response, and this study lays the foundation for exploring the functions of bZIP and provides a theoretical basis for improving the drought tolerance of D. catenatum.

1. Introduction

Plants are subjected to a series of stresses during their life cycle. Transcription factors (TFs) play key roles in regulating biological processes in plant cells. When plants are stimulated by cold, heat, salt or drought stresses, a series of signal transductions can induce TFs to specifically bind to cis-acting elements and regulate the transcription expression of stress responsive genes, thereby improve plant stress resistance [1,2]. More than 60 TF families have been identified in plants [3]. As one of the largest TF families in plants [4,5], basic leucine zipper (bZIP) plays a critical regulatory role in plant development and growth, pest defense, abiotic stress and other physiological processes [6,7,8].
The bZIP transcription factor contains two conserved domains consisting of about 60~80 amino acid residues: one is the highly conserved alkaline amino acid region at the N-terminal, consisting of about 16~20 amino acid residues, which acts as a nuclear localization signal by binding to specific DNA sequences via a N-X7-R/K structure [9]; the other is the low conservative leucine zipper region at the C-terminal [10], which is composed of several repeated heptapeptides or hydrophobic amino acid residues and is mainly responsible for the heterogenic or homologous dimerization of bZIP protein before binding to DNA [11,12].
Many eukaryotes have the bZIP TF family, for example, 89 bZIP TFs have been identified in rice [13], 75 in Arabidopsis thaliana [5], 55 in grape [14], 64 in cucumber [15], 160 in soybean [16], 66 in Iponoea trifida [17], 97 in oil palm [18] and 84 in pear [19]. Different numbers of bZIP gene families lead to functional changes in response to different kinds of biotic and abiotic stresses [20]. Previous studies have shown that the rice OsbZIP23 functions in the ABA signaling pathway, and the transgenic plants exhibited enhanced tolerance to drought stress [21]. OsbZIP71 was also found to enhance drought tolerance in an ABA-mediated pathway in rice [22]. Furthermore, OsbZIP71 formed heterodimers with OsbZIP73, and increased cold tolerance in the rice reproductive stage [23]. Arabidopsis and soybean overexpression of GmbZIP2 improved plant resistance to drought and salt stresses [24], while GmbZIP15 negatively regulated salt and drought tolerance in soybean [25]. Under drought stress, 16 MdbZIP genes were differentially expressed in apple roots and leaves, indicating that these MdbZIP genes were associated with drought stress response [26]. The Vigna radiata bZIP transcription factor VrbZIP52 was significantly upregulated after polyethylene glycol (PEG) treatment, indicative of the involvement of VrbZIP52 in drought stress response [27]. After treatment with PEG, 8627 and 9281 differentially expressed genes (DEGs) were detected in the Boehmeria nivea roots and leaves, respectively, among which the bZIP transcription factors Comp28477 and Comp58004 may be related to drought stress [28]. After PEG treatment, the expression of 10 CsbZIPs were upregulated in cucumber roots and downregulated in leaves, indicative of tissue-specific expression of the bZIP gene [15].
Dendrobium catenatum is a perennial herb of the Orchid family, and has a long history in edible flowers. It is usually eaten fresh, as a flower dish or dried and dehydrated into a tea drink. Importantly, D. catenatum contains many active ingredients such as Dendrobium polysaccharide, which can scavenge free radicals in the body and improve immunity. However, wild D. catenatum is mostly attached to cliffs and rock crevices in high mountains; therefore, it often faces an adversity threat, especially of periodic drought [29,30], making wild resources scarce. Bionic cultivations are the main modes of D. catenatum artificial cultivation and these methods can affect the yield and quality of D. catenatum. The yield of the dry bark cultivation method was found to be highest, while the contents of dendrobium and polysaccharide were highest in the living trunk cultivation mode [31]. Therefore, identifying drought stress-related genes in the D. catenatum genome and investigating their functions is necessary and helpful. The genome-wide identification of some TF families in D. catenatum has been conducted and reported, such as MYB [32], WRKY [33] and NAC [34]. However, the bZIP gene family in the plant has not been investigated. In this study, based on the whole genome sequence of D. catenatum [35], we identified the bZIP gene family and analyzed their characteristics using bioinformatics methods. The expression of bZIP gene members under drought stress was further analyzed using RT-qPCR based on RNA-seq data. This study provides a theoretical basis for further improving the drought resistance of D. catenatum.

2. Materials and Methods

2.1. Identification of the bZIP Family Members of D. catenatum

The protein sequence, genome sequence, coding sequence (CDS) and GFF annotation files of D. catenatum were obtained from the NCBI GenBank database (PRJNA262478) [35]. To identify the bZIP gene family of D. catenatum, the bZIP domain (PF00170) hidden Markov model was acquired from the Pfam database (http://pfam.xfam.org/, assessed on 16 September 2021) and the database of D. catenatum proteins was queried through the Bio-linux bioinformatics documentation system. The conservative bZIP domains were detected by screening and submitting all the output proteins with E values ≤ 1 × 10−10 [36] to the Simple Modular Architecture Research Tool (SMART, v9; http://smart.embl.de/smart/batch.pl, assessed on 16 September 2021) database and the Conserved Domain Database (CDD, v3.19; https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/cdd/, assessed on 16 September 2021). The DcbZIP proteins were obtained after removal of incomplete and redundant sequences. Characteristics including the molecular weight (MW), the isoelectric point (pI) and amino acid number of the proteins were determined using the tool ExPASy (https://web.expasy.org/protparam/, assessed on 22 April 2022). The PSORT tool (https://www.genscript.com/psort.html, assessed on 22 April 2022) was applied to predict the subcellular localizations of the proteins.

2.2. Phylogenetic Analysis of bZIP Proteins

The bZIP proteins in A. thaliana and rice were identified as described previously [5,13]. To explore the phylogenetic relationships between the bZIP proteins, the protein sequences from A. thaliana, rice and D. catenatum (Gene IDs are shown in Supplementary Table S1) were aligned with clustalw 2.0, and the maximum likelihood (ML) phylogenetic tree was constructed using MEGA 6.0 software with default parameters, and was modified and visualized as described previously [34].

2.3. Analysis of Gene Structure, Conserved Motifs, and Promoter Cis-Regulatory Elements

The MEME (https://meme-suite.org/meme/doc/meme.html, assessed on 26 September 2022) online tool was used to predict the conserved motifs of the D. catenatum bZIP proteins with parameters of optimum motif width of 6~50 amino acid residues and maximum number of motifs of 20 [37]. TBtools software [38] was used to extract the bZIP gene structure file from the gff annotation file of D. catenatum genome, to extract the 2000 bp upstream sequence of the start codon (ATG) of D. catenatum bZIP gene as promoter sequence and to visualize the conserved motifs, gene structures and cis-elements following detection of cis-elements using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, assessed on 22 September 2022) software. Finally, the conserved motifs, gene structures and the cis-elements were visualized by TBtools.

2.4. Spatial Expression Profile Analysis Using RNA-Seq Data

The raw RNA-seq reads of different tissues (SRP091756) were obtained from the sequence read archive (SRA) database (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/sra, assessed on 12 April 2022). The gene expression data of different tissues (stem, root, root tip, white root, leaf, lip, sepal, column, flower bud and pollinia) were calculated and normalized to the transcripts per million (TPM) values by kallisto tools [34,39]. In this study, we screened the expression data of bZIP genes using their gene IDs from the normalized TPM values. A heatmap of differential expression of bZIP genes was constructed using TBtools.

2.5. Stress Treatments

D. catenatum seeds (D. catenatum ‘Guangnan’) were collected from Guangnan country, Yunnan province, China. The seeds were sterilized and used as explants to induce protocorm. After proliferation and regeneration, the complete plants were induced. The D. catenatum tissue culture seedlings were obtained through protocorm induction and plant regeneration from the mother plants, and then used in this study. The experiment was carried out in completely randomized design and conducted in the greenhouse in Hainan University. In the present study, the three-month-old tissue culture seedlings were grown in 1/2 MS medium in a growth chamber under the conditions as follows: light for 12 h, 70% humidity and temperature of 22–25 °C [32]. The uniform and robust growing seedlings were selected and treated with 20% PEG8000 for drought stress [32]. After treatment for 3, 6, 9, 12, 24 or 48 h, the stems, leaves and roots were randomly collected from five seedlings, mixed, quickly frozen in liquid nitrogen and stored at −80 °C for RNA extraction and gene expression analysis [32].

2.6. Gene Expression Analysis under Drought Treatment

The RNA seq data (SRP132541) of the D. catenatum leaves collected from plants treated with different drought stresses with a base matrix water content of 0%, 10–15% and 30–35% were downloaded from the public SRA database according to a previous study [40] and their expression profiles of DcbZIP genes were analyzed. Then, the TPM values were used for heatmap construction with TBtools. The differential genes were aggregated according to T1 and T2 values (T1 and T2 equal to the TPM of 10–15% and 0% water content, respectively, divided by a TPM of 30–35% water content).
RNA extraction and cDNA synthesis were performed according to a previous study [32]. Real-time fluorescence quantitative PCR (RT-qPCR) was used for analysis of samples treated with PEG as described by Zhang et al. [32] with the primers shown in Supplemental Table S2.

2.7. Analysis of Co-Expression and Protein–Protein Interaction Networks of DcbZIP Proteins

According to the TPM value of RNA-seq data, the DcbZIP gene co-expression relationships were analyzed using Origin software (version 2022b) [41]. The co-expression network analysis was performed using Cytoscape (version 3.9.1) based on their correlation coefficients [42]. The data used for correlation analysis were the means of four replications. The interactions between bZIP homolog proteins and other proteins were analyzed using STRING software (https://string-db.org/, assessed on 20 October 2022) [43].

3. Results

3.1. Identification and Physicochemical Properties of bZIP Family in D. catenatum

In total, 62 bZIP genes were identified from the D. catenatum genome by the bioinformatics method. The bZIP family members were named as DcbZIP1 to DcbZIP62 based on their relationships with rice and A. thaliana bZIP proteins (Table 1). The proteins encoded by the bZIP gene had an amino acid number ranging from 130 to 692, a molecular weight (MW) ranging from 15.24 to 74.94 kDa and a theoretical isoelectric point (pI) ranging from 5.13 (DcbZIP60) to 11.58 (DcbZIP5). DcbZIP8 and DcbZIP58 encoded proteins with the fewest amino acids and smallest MWs, and DcbZIP28 encoded a protein with the most amino acids with the largest MW. Most DcbZIP proteins were predicted to localize in the cell nucleus, while DcbZIP15 was localized in cytoplasm, and DcbZIP11 and DcbZIP14 were localized in mitochondria.

3.2. Phylogenetic Analysis of bZIP Proteins

An ML phylogenetic tree was constructed to study the evolutionary relationships of bZIP proteins using the bZIP family protein sequences from D. catenatum (Dc), A. thaliana (At) and rice (Os) (Supplementary Table S1). The bZIP proteins could be divided into 10 subgroups, as shown in Figure 1, consistent with those in rice and Arabidopsis [5,44]. The D. catenatum bZIP family members were distributed unequally in the subgroups (Figure 1 and Table 1). The subgroup S contained the most members, with fifteen bZIP genes (24.20% of the whole gene family), followed by subgroup A with eleven bZIP genes (17.74%), subgroup D with ten bZIP genes (16.13%), subgroup I with nine bZIP genes (14.51%), subgroup G with four bZIP genes (6.45%), subgroups B, C, E and H, which contained three bZIP genes (4.83%) and subgroup F, which only contained one bZIP gene (1.61%). The gene distribution of each subgroup in D. catenatum was similar to that of rice and A. thaliana (Supplementary Figure S1), suggesting that in these three species, the evolution trajectory of the bZIP gene was similar.

3.3. Conserved Motif and Gene Structure of bZIP Family Genes

The bZIP family of D. catenatum can be divided into 10 subgroups according to the evolutionary analysis (Figure 1 and Figure 2A). The conserved motifs in the sequences of these proteins were then predicted using MEME software to elucidate their diversity and conservation (Figure 2B), and 20 motifs were identified (Supplementary Figure S2). Different subgroups contained different types and numbers of conserved motifs, while most of the DcbZIP proteins within the same subgroup displayed similar motif compositions (Supplementary Table S3). Motifs 8 and 17 were specific to subgroup I, whereas motif 20 was unique to subgroup C. Almost all the proteins contained motif 1, which was annotated as the bZIP domain, including two domains: a leucine zipper region and a basic region. The leucine zipper region consisted of 15 amino acid residues with an amino acid sequence model of L-X6-L-X6-L, and the basic region consisted of 18 amino acid residues with an amino acid sequence model of N-X7-R-X9. The leucine in the leucine zipper region was not conserved and could be replaced by methionine (M), valine (V), phenylalanine (F) and isoleucine (I), all of which are hydrophobic amino acids (Supplementary Figure S3).
Gene structure analysis showed that the number of introns in the DcbZIP gene family ranged from 0 to 12 (Figure 2C and Supplementary Table S3). Approximately 80.65% of the DcbZIP genes contained fewer than five introns. The subgroup D members, except DcbZIP23, contained more than eight introns. DcbZIP16, DcbZIP20 and DcbZIP21 had the most numbers of introns (12 introns). Meanwhile, the same subgroup of DcbZIP genes had similar intron/exon structures. For instance, the subgroup S DcbZIP genes contained 0–3 introns, among which 73.33% (11/15) had no intron. Both subgroups H and E contained three introns.

3.4. Cis-Element Analysis of bZIP Gene Promoters in D. catenatum

The upstream 2000 bp sequence of the DcbZIP gene start codon was extracted by TBtools software as promoters, and the cis-acting elements were then analyzed using PlantCARE online software (Figure 3 and Supplementary Table S4). Results revealed that the 21 cis-acting elements were mainly divided into four categories: stress-related elements, light-responsive elements, plant growth and development-related elements and hormone response elements (Figure 3B,C). Plant hormone response elements mainly included ABRE elements involved in abscisic acid regulation, salicylic acid-responsive elements (SARE and TCA element), gibberellin-responsive elements (TATC-box, GARE-motif and P-box) and auxin-responsive elements (TGA element and AuxRR-core). Among these elements, SARE only existed in the DcbZIP40 and DcbZIP61 promoter regions, suggesting that these two genes might be involved in the SA signaling pathway. Plant growth and development related elements mainly included circadian elements, seed-specific regulatory element (RY-element), meristem expression-related element (CAT-box), endosperm expression element (GCN4_motif) and zein metabolic regulatory element (O2-site). Light responsive elements mainly included the GT1-motif and MRE elements. Stress-related elements included wound-responsive element (WUN-motif), low-temperature responsiveness (LTR), drought-inducibility (MBS) and stress and defense responsiveness (TC-rich repeats). Among these elements, LTR was detected in 35 (56%) DcbZIP gene promoters, MBS was detected in 27 (44%) DcbZIP gene promoters, WUN-motif existed in 7 (11%) DcbZIP gene promoters, and TC-rich repeat elements existed in 24 (39%) DcbZIP gene promoters. In addition, 33 (53%) gene promoters were observed to contain more than two stress-related elements (Figure 3C), suggesting that DcbZIP may be widely involved in abiotic stress response.

3.5. Tissue-Specific Expression Profile of DcbZIP Gene Based on RNA-Seq

The DcbZIP gene expression levels in different tissues were detected according to the RNA-seq data from the public SRA database (SRP091756) (Supplementary Table S5). Based on the TPM values of DcbZIP genes in 10 different tissues including stem, root, leaf, lip, root tip, sepal, white root, flower bud, column and pollinia, their spatial expression patterns were displayed using a heatmap, as shown in Figure 4. The DcbZIP genes showed different expression patterns. For example, 10 genes (DcbZIP10, DcbZIP11, DcbZIP14, DcbZIP15, DcbZIP16, DcbZIP17, DcbZIP26, DcbZIP28, DcbZIP30 and DcbZIP55) were highly expressed in the roots, 13 genes (DcbZIP3, DcbZIP7, DcbZIP8, DcbZIP20, DcbZIP27, DcbZIP33, DcbZIP38, DcbZIP41, DcbZIP42, DcbZIP47, DcbZIP51, DcbZIP57 and DcbZIP62) were highly expressed in stems, 3 genes (DcbZIP25, DcbZIP38 and DcbZIP58) were highly expressed in leaves, 11 genes (DcbZIP1, DcbZIP4, DcbZIP5, DcbZIP6, DcbZIP8, DcbZIP9, DcbZIP42, DcbZIP48, DcbZIP53, DcbZIP56 and DcbZIP58) showed high expression levels in sepals, 5 genes (DcbZIP1, DcbZIP4, DcbZIP9, DcbZIP37 and DcbZIP40) were highly expressed in lips, 14 genes (DcbZIP2, DcbZIP5, DcbZIP13, DcbZIP17, DcbZIP18, DcbZIP19, DcbZIP24, DcbZIP32, DcbZIP34, DcbZIP36, DcbZIP39, DcbZIP52, DcbZIP59 and DcbZIP61) showed high expression levels in pollinia, 5 genes (DcbZIP16, DcbZIP28, DcbZIP46, DcbZIP59 and DcbZIP61) were highly expressed in flower buds and 7 genes (DcbZIP6, DcbZIP29, DcbZIP30, DcbZIP42, DcbZIP44, DcbZIP48 and DcbZIP54) were highly expressed in columns. These results indicate tissue-specific expression of DcbZIP genes, suggesting that these genes might be involved in growth and development of different tissues.

3.6. DcbZIP Gene Expression under Drought Stress Based on RNA-Seq

The drought RNA-seq data of D. catenatum leaf samples (SRP132541) were obtained from the public SRA database according to a previous study [40], and the TPM values of DcbZIP genes were calculated to study the response of DcbZIP genes to drought stress (Supplementary Table S6). When the matrix water content decreased from 30–35% to 0%, different expression patterns were observed for different DcbZIP genes (Figure 5), indicating that these DcbZIP genes were highly sensitive to drought stress. The expression patterns of 62 DcbZIP genes in D. catenatum under drought stress were shown in a hierarchical clustering heat map. Based on their expression patterns, these genes were classified into four clusters. In cluster I, there were two genes with T1 and T2 values >2, suggesting they might function in drought stress response. Thirteen DcbZIP genes were grouped into cluster II with T1 and T2 values <1, indicating their decreased expression with increasing drought stress. The T1 and T2 values were both more than one for all members in cluster III, except for DcbZIP58 (0.52), DcbZIP28 (0.32), DcbZIP34 (0.71), DcbZIP16 (0.62), DcbZIP30 (0.98) and DcbZIP45 (0.98). The T2 value of most genes was greater than the T1 value, indicating that the expression levels of genes under 0% treatment were higher than those under 10–15% treatment, suggesting that most cluster III genes might play critical roles in drought stress response. In cluster IV, seven of the fourteen DcbZIPs had T1 and T2 values <1. In total, 33 DcbZIP genes in clusters I and III might be involved in positive regulation of D. catenatum drought tolerance, so they were selected for further study.

3.7. Analysis of Drought-Related DcbZIP Genes

Co-expression analysis has been broadly used to screen key genes from large gene members [45]. In order to explore the relationships of drought-related bZIP genes, the 33 genes in clusters I and III DcbZIP with T1 and T2 values >1 were subjected to correlation analysis using Origin software. Genes with absolute Pearson correlation coefficients of >0.95 were selected. The results revealed that 140 pairs of DcbZIP genes were positively correlated and 78 pairs of DcbZIP genes were negatively correlated (Figure 6A). A co-expression network was constructed using Cytoscape based on Pearson correlation coefficients (Figure 6B). A total of 17 core genes (DcbZIP24, DcbZIP45, DcbZIP53, DcbZIP12, DcbZIP28, DcbZIP47, DcbZIP61, DcbZIP58, DcbZIP23, DcbZIP30, DcbZIP8, DcbZIP60, DcbZIP34, DcbZIP9, DcbZIP6, DcbZIP3 and DcbZIP42) were identified, among which the DcbZIP12, DcbZIP24, DcbZIP45 and DcbZIP53 genes dominated in the network due to their high connectivity with other genes.
To further investigate the functions of these 17 core genes, the interaction network between these DcbZIP proteins and their homologous AtbZIP proteins in Arabidopsis was predicted by STRING software. A total of 34 interacting proteins were predicted and their annotated information is shown in Supplemental Table S7. As shown in Figure 7, AFP3 (involved in ABA signaling) and DPBF2 (involved in ABA signaling) interacted with AREB3 (homologous protein of DcbZIP24). Furthermore, AREB3 also had associations with SNRK2.2/2.3/2.4/2.10, OST1 and BZIP34/6. Arabidopsis AHBP-1B, homologous to DcbZIP45, could interact with NPR1. EEL (homologue of DcbZIP12) had associations with OST1, bZIP1, BZIP34, BZIP60 and TGA10. BZIP53, homologous to DcbZIP53, DcbZIP58 and DcbZIP8, could interact with BZIP25, bZIP1, bZIP44, BZIP17, NAC089 and TGA1. AT4G38900 (homologue of DcbZIP61 and DcbZIP30) had an association with TGA10. bZIP19 (homologue of DcbZIP23) could interact with BZIP34 and bZIP68. The proteins interacting with bZIP6 (homologous to DcbZIP6) had associations with bZIP1, bZIP44 and bZIP68. BZIP60 (homologous to DcbZIP60) had associations with EEL, BZIP17, IRE1-1, BZIP53 and bZIP1. BZIP9 (homologous to DcbZIP9) could interact with bZIP1, BZIP53, BZIP34 and BZIP60. bZIP44, the homologue of DcbZIP3, had associations with BZIP25, AT4G38900, TGA10, BZIP60, BZIP53, bZIP1, ERD5 and BZIP17. BZIP17 (homologue of DcbZIP28) had associations with BZIP60, NAC089, IRE1-1, BZIP34, bZIP1 and BZIP53. TGA1 (homologue of DcbZIP47) could interact with NPR1, NPR3 and NPR4. BZIP34, the homologue of DcbZIP34, had associations with EEL, TGA10 and bZIP1.

3.8. Expression Profile Analysis of 17 Core DcbZIP Genes under Drought Stress Using RT-qPCR

The expression levels of 17 core DcbZIP genes under 20% PEG8000 treatment were analyzed using RT-qPCR to investigate their response to drought stress. As shown in Figure 8, consistent with the RNA-seq data, after PEG treatment, the expression levels of most DcbZIP genes increased. The DcbZIP34 and DcbZIP42 genes in roots were significantly increased and showed the highest expression level after 3-h and 48-h treatments, with a 15-fold and 18-fold increase, respectively, compared to 0 h. The DcbZIP6 and DcbZIP47 genes in stems were highly induced after drought stress treatment, with 133-fold and 130-fold higher expression levels compared with the control, respectively. However, the increasing trend in DcbZIP genes was not obvious in leaves.

4. Discussion

The bZIP TF is widely involved in the regulation of plant development, growth and response to biotic and abiotic stresses [46,47,48], which has been identified and analyzed in a variety of plants such as Arabidopsis [5], sorghum [49], barley [50], maize [51], rice [44], soybean [16] and rape [52]. However, there are few reports on the bZIP family genes of D. catenatum. Sixty-two bZIP gene family members were identified using bioinformatics techniques from the D. catenatum genome in this study. Similar gene numbers have been reported in other plants. Isatis indigotica has 65 bZIP genes [53], Pyrus bretschneideri has 62 PbbZIP genes [54], cucumber has 64 bZIP genes [15] and watermelon has 62 ClabZIP genes [55]. The phylogenetic relationships of bZIP proteins and their response to drought stress, conserved motif, promoter region cis-acting elements and gene structure were analyzed.
The bZIP gene family can be classified into 10 subgroups based on phylogenetic analysis (Figure 1), with subgroup F containing the fewest members and subgroup S containing the most members. The subgroup proportions were similar to those in rice and Arabidopsis (Supplementary Figure S1), as well as in switchgrass [56] and mung bean [57], indicating evolutionary conservation of the bZIP gene in plants. Previous studies showed that the subgroup A proteins AtbZIP39, AtbZIP36, AtbZIP38 and AtbZIP35 played important roles in the response to stress and ABA treatment [58,59,60], suggesting the same subgroup of proteins has a similar function. In addition, AtbZIP10 and AtbZIP25 in subgroup C could regulate the expression of storage proteins in A. thaliana [5]. Subgroup D members functioned by preventing pathogen invasion and plant development [5]. Subgroup G proteins were involved in UV and blue light signal transduction [61]. AtbZIP56 in subgroup H functioned in the development of photomorphology in seedlings [62]. Therefore, we could speculate that the D. catenatum genes in the same group may have similar functions. Although phylogenetic analysis provided important information for understanding the functions of candidate genes, their functions cannot be clearly characterized based on the phylogenetic analysis alone. To this end, we examined additional evidence including gene structures and conserved motifs to support the reliable subgroup classification.
Gene structure is a critical basis for gene evolution and gene amplification studies [56]. Analysis of structure features of the bZIP gene family in D. catenatum, showed that the presence of introns and exons in each subgroup exhibited similar rules. The DcbZIP genes in subgroup S contained 0–3 introns, among which 73.33% (11/15) of members had no introns. Both subgroups H and E of DcbZIP genes contained three introns (Figure 2C and Supplementary Table S3), similar to those found in Chinese jujube [63], soybean [16], switchgrass [56] and grape [14], suggesting the intron-exon structure was conserved. It should be noted that some DcbZIP genes, especially those in the subgroup S, generally lacked introns (Figure 2C and Supplementary Table S3), which may shorten the post-transcriptional process. Thus, they can immediately respond to abiotic stress [64]. Similarly, the subgroup S of bZIP genes in soybeans and watermelons also lacked introns [16,55]. Genes lacking introns evolve faster than intron acquisition after gene amplification, and most bZIP genes in subgroups D and G had more introns than those in other subgroups (Figure 3). Therefore, the subgroups D and G, members of D. catenatum bZIP gene family, may contain more original genes compared with other subgroups [65]. In addition, 20 motifs were predicted in DcbZIP proteins using MEME analysis. The different conserved motif number and composition in different subgroups indicated their functional differences, which is important for determining specific functions of bZIP proteins [66].
The promoter region cis-acting elements can bind to target genes and induce their expression under abiotic stresses [67,68]. Arabidopsis AtbZIP17 has a high homology with tobacco TGA1b protein, which belongs to the TGA/OBF subfamily of bZIP TFs. Both AtbZIP17 and TGA1b can specifically bind to TGACG elements to regulate downstream gene expression [69]. All the promoters of 51 tomato HD-Zip genes contain the low temperature-responsive element (LTR), indicating their involvement in plant response to low temperature stress [70]. The promoter of D. catenatum, bZIP transcription factor, contained multiple hormone signaling-related and stress response-related elements (Figure 3), such as ABRE, SARE, LTR and MBS. The results indicated that DcbZIP transcription factor was widely involved in the regulation of plant stress response. Among them, 35 DcbZIP gene promoters contained LTR elements and 27 DcbZIP gene promoters contained MBS elements, suggesting that most bZIP genes in D. catenatum were related to abiotic stress.
Wild D. catenatum grows in cliffs and rock crevices and is often threatened by adversity. Drought stress is the main affecting factor for its growth and development. The ABA signaling pathway plays an important role in plant response to abiotic stress [71,72]; AREB3 (homologous protein of DcbZIP24), as an ABA-responsive element-binding protein (AREB) [73], plays a key role in gene expression regulation involved in ABA signaling [68]. Meanwhile, the interaction network displayed that AREB3 could interact with BZIP34, a homologue protein of DcbZIP34. These results suggested that DcbZIP24 and DcbZIP34 might regulate drought resistance by affecting ABA signal transduction pathways. Overexpression of AtbZIP1 improved plant tolerance to salt, osmotic and drought stress [74]. AtbZIP1 is an interacting protein of bZIP6 (homologue protein of DcbZIP6). The results indicated that DcbZIP6 may be involved in regulating plant drought tolerance. The above analysis suggested that many DcbZIPs might play key roles in the response to drought stress. Therefore, the expression of DcbZIP genes under drought stress was studied next. The DcbZIP gene expression under different drought conditions was first analyzed using public RNA-seq data, and these genes were divided into four clusters according to their expression levels. Among them, 33 DcbZIP genes belonging to clusters I and III were upregulated under different drought treatments, and correlation analysis showed that most of the upregulated genes were positively related (Figure 6A), indicating that they may have a synergistic effect in development of drought resistance. In addition, 17 core genes that were closely correlated with other genes were screened out using a co-expression network (Figure 7B). These results suggest that these 17 key genes may play leading roles in the development of drought resistance. RT-qPCR analysis further revealed that DcbZIP6 and DcbZIP47 genes in stems and DcbZIP34 and DcbZIP42 genes in roots were significantly upregulated under drought stress (Figure 8), suggesting these four genes can significantly contribute to the development of drought resistance in D. catenatum, and more research on their functions might be performed by transgenic or CRISPR techniques in the future.

5. Conclusions

In this study, 62 DcbZIP TFs were identified in the D. catenatum genome, which were divided into 10 subgroups based on their correlations with rice and Arabidopsis bZIP proteins. Gene structures and conserved motifs indicated similarities in bZIP clustering in the phylogenetic tree. Promoter cis-element analysis indicated the potential multiple roles of bZIP in abiotic stresses. Using the public drought RNA-seq data, the DcbZIP genes were classified into four clusters based on their expression levels, among which 17 DcbZIP genes were further screened by RT-qPCR and four DcbZIP genes (DcbZIP6, DcbZIP34, DcbZIP42 and DcbZIP47) were finally found to be highly induced by drought stress, which could be main contributors to drought tolerance in D. catenatum. For the first time, this study performed a genome-wide analysis of the DcbZIP gene family in D. catenatum and investigated their expression patterns under drought stress. This study provides a foundation for the molecular breeding of drought tolerance in D. catenatum.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agronomy13010236/s1, Figure S1: The percentage of members in each bZIP subgroup in D. catenatum (A), A. thaliana (B) and O. sativa (C); Figure S2: The amino acid sequences of each motif identified in DcbZIP proteins; Figure S3: Sequence alignment of motif 1 in DcbZIP proteins; Table S1: The gene ID of bZIP transcription factors in D. catenatum, A. thaliana and O. sativa; Table S2: Primers used in this study; Table S3: Motifs and the number of introns of each DcbZIP gene; Table S4: Information on cis-elements of DcbZIP gene promoters; Table S5: Expression of DcbZIP genes in different tissues (TPM); Table S6: Expression of DcbZIP genes under drought stress (TPM); Table S7: Annotation information of DcbZIP homologous proteins.

Author Contributions

Conceptualization, W.Y. and Y.Z.; formal analysis, P.W., Y.L., T.Z., Y.K., W.L., J.W., W.Y. and Y.Z.; funding acquisition, W.Y. and Y.Z.; investigation, P.W., Y.L., T.Z. and Y.Z.; methodology, P.W. and Y.L.; supervision, Y.Z; writing—original draft preparation, P.W., Y.L., W.Y. and Y.Z.; writing—review and editing, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 31660580 by W.Y.), the Hainan Provincial Natural Science Foundation of China (No. 319MS009 by Y.Z.), the State Key Laboratory of Cotton Biology Open Fund (CB2021A16 by Y.Z.), the Education Department of Hainan Province (No. Hnky2021-19 by Y.Z.), the Opening Project Fund of Key Laboratory of Biology and Genetic Resources of Rubber Tree and the Ministry of Agriculture and Rural Affairs (No. RRI-KLOF202003 by W.Y.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Izawa, T.; Foster, R.; Chua, N.H. Plant bZIP protein DNA binding specificity. J. Mol. Biol. 1993, 230, 1131–1144. [Google Scholar] [CrossRef] [PubMed]
  2. Kasuga, M.; Liu, Q.; Miura, S.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol. 1999, 17, 287–291. [Google Scholar] [CrossRef] [PubMed]
  3. Pérez-Rodríguez, P.; Riaño-Pachón, D.M.; Corrêa, L.G.; Rensing, S.A.; Kersten, B.; Mueller-Roeber, B. PlnTFDB: Updated content and new features of the plant transcription factor database. Nucleic Acids Res. 2010, 38, D822–D827. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Riechmann, J.L.; Heard, J.; Martin, G.; Reuber, L.; Jiang, C.; Keddie, J.; Adam, L.; Pineda, O.; Ratcliffe, O.J.; Samaha, R.R.; et al. Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science 2000, 290, 2105–2110. [Google Scholar] [CrossRef] [PubMed]
  5. Jakoby, M.; Weisshaar, B.; Dröge-Laser, W.; Vicente-Carbajosa, J.; Tiedemann, J.; Kroj, T.; Parcy, F. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 2002, 7, 106–111. [Google Scholar] [CrossRef] [PubMed]
  6. Hsieh, W.P.; Hsieh, H.L.; Wu, S.H. Arabidopsis bZIP16 transcription factor integrates light and hormone signaling pathways to regulate early seedling development. Plant Cell 2012, 24, 3997–4011. [Google Scholar] [CrossRef] [Green Version]
  7. Yoshida, T.; Fujita, Y.; Sayama, H.; Kidokoro, S.; Maruyama, K.; Mizoi, J.; Shinozaki, K.; Yamaguchi-Shinozaki, K. AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. Plant J. 2010, 61, 672–685. [Google Scholar] [CrossRef]
  8. Kobayashi, F.; Maeta, E.; Terashima, A.; Takumi, S. Positive role of a wheat HvABI5 ortholog in abiotic stress response of seedlings. Physiol. Plant 2008, 134, 74–86. [Google Scholar] [CrossRef] [Green Version]
  9. Lee, S.C.; Choi, H.W.; Hwang, I.S.; Choi, D.S.; Hwang, B.K. Functional roles of the pepper pathogen-induced bZIP transcription factor, CAbZIP1, in enhanced resistance to pathogen infection and environmental stresses. Planta 2006, 224, 1209–1225. [Google Scholar] [CrossRef] [PubMed]
  10. Leite, J.P.; Barbosa, E.G.; Marin, S.R.; Marinho, J.P.; Carvalho, J.F.; Pagliarini, R.F.; Cruz, A.S.; Oliveira, M.C.; Farias, J.R.; Neumaier, N.; et al. Overexpression of the activated form of the AtAREB1 gene (AtAREB1ΔQT) improves soybean responses to water deficit. Genet. Mol. Res. 2014, 13, 6272–6286. [Google Scholar] [CrossRef]
  11. Landschulz, W.H.; Johnson, P.F.; McKnight, S.L. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science 1988, 240, 1759–1764. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Ellenberger, T.E.; Brandl, C.J.; Struhl, K.; Harrison, S.C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. Cell 1992, 71, 1223–1237. [Google Scholar] [CrossRef] [PubMed]
  13. Corrêa, L.G.; Riaño-Pachón, D.M.; Schrago, C.G.; dos Santos, R.V.; Mueller-Roeber, B.; Vincentz, M. The role of bZIP transcription factors in green plant evolution: Adaptive features emerging from four founder genes. PLoS ONE 2008, 3, e2944. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, J.; Chen, N.; Chen, F.; Cai, B.; Santo, S.D.; Tornielli, G.B.; Pezzotti, M.; Cheng, Z.M. Genome-wide analysis and expression profile of the bZIP transcription factor gene family in grapevine (Vitis vinifera). BMC Genom. 2014, 13, 281. [Google Scholar] [CrossRef] [Green Version]
  15. Baloglu, M.C.; Eldem, V.; Hajyzadeh, M.; Unver, T. Genome-wide analysis of the bZIP transcription factors in cucumber. PLoS ONE 2014, 9, e96014. [Google Scholar] [CrossRef] [Green Version]
  16. Zhang, M.; Liu, Y.; Shi, H.; Guo, M.; Chai, M.; He, Q.; Yan, M.; Cao, D.; Zhao, L.; Cai, H.; et al. Evolutionary and expression analyses of soybean basic Leucine zipper transcription factor family. BMC Genom. 2018, 19, 159. [Google Scholar] [CrossRef] [Green Version]
  17. Yang, Z.; Sun, J.; Chen, Y.; Zhu, P.; Zhang, L.; Wu, S.; Ma, D.; Cao, Q.; Li, Z.; Xu, T. Genome-wide identification, structural and gene expression analysis of the bZIP transcription factor family in sweet potato wild relative Ipomoea trifida. BMC Genet. 2019, 20, 41. [Google Scholar] [CrossRef] [Green Version]
  18. Zhou, L.; Yarra, R. Genome-wide identification and expression analysis of bZIP transcription factors in oil palm (Elaeis guineensis Jacq.) under abiotic stress. Protoplasma 2022, 259, 469–483. [Google Scholar] [CrossRef]
  19. Liu, L.; Zhang, Y.X.; Wang, Q.; Tao, X.Y.; Fang, J.; Zheng, W.J.; Zhu, L.W.; Jia, B.; Heng, W.; Li, S.W. Identification of bZIP transcription factors and their responses to brown spot in pear. Genet. Mol. Biol. 2022, 45, e20210175. [Google Scholar] [CrossRef]
  20. Li, Q.; Yu, H.; Cao, P.B.; Fawal, N.; Mathé, C.; Azar, S.; Cassan-Wang, H.; Myburg, A.A.; Grima-Pettenati, J.; Marque, C.; et al. Explosive tandem and segmental duplications of multigenic families in Eucalyptus grandis. Genome Biol. Evol. 2015, 7, 1068–1081. [Google Scholar] [CrossRef]
  21. Zong, W.; Tang, N.; Yang, J.; Peng, L.; Ma, S.; Xu, Y.; Li, G.; Xiong, L. Feedback regulation of ABA signaling and biosynthesis by a bZIP transcription factor targets drought-resistance-related genes. Plant Physiol. 2016, 171, 2810–2825. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Liu, C.; Mao, B.; Ou, S.; Wang, W.; Liu, L.; Wu, Y.; Chu, C.; Wang, X. OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice. Plant Mol. Biol. 2014, 84, 19–36. [Google Scholar] [CrossRef] [PubMed]
  23. Liu, C.; Schlappi, M.R.; Mao, B.; Wang, W.; Wang, A.; Chu, C.C. The bZIP73 transcription factor controls rice cold tolerance at the reproductive stage. Plant Biotech. J. 2019, 17, 1834–1849. [Google Scholar] [CrossRef] [Green Version]
  24. Yang, Y.; Yu, T.F.; Ma, J.; Chen, J.; Zhou, Y.B.; Chen, M.; Ma, Y.Z.; Wei, W.L.; Xu, Z.S. The soybean bZIP transcription factor gene GmbZIP2 confers drought and salt resistances in transgenic plants. Int. J. Mol. Sci. 2020, 21, 670. [Google Scholar] [CrossRef] [Green Version]
  25. Zhang, M.; Liu, Y.; Cai, H.; Guo, M.; Chai, M.; She, Z.; Ye, L.; Cheng, Y.; Wang, B.; Qin, Y. The bZIP transcription factor GmbZIP15 negatively regulates salt- and drought-stress responses in soybean. Int. J. Mol. Sci. 2020, 21, 7778. [Google Scholar] [CrossRef]
  26. Li, Y.Y.; Meng, D.; Li, M.J.; Cheng, L.I. Genome-wide identification and expression analysis of the bZIP gene family in apple (Malus domestica). Tree Genet. Genomes 2016, 12, 82. [Google Scholar] [CrossRef]
  27. Wang, L.; Zhu, J.; Li, X.; Wang, S.; Wu, J. Salt and drought stress and ABA responses related to bZIP genes from V. radiata and V. angularis. Gene 2018, 651, 152–160. [Google Scholar] [CrossRef]
  28. An, X.; Chen, J.; Zhang, J.; Liao, Y.; Dai, L.; Wang, B.; Liu, L.; Peng, D. Transcriptome profiling and identification of transcription factors in ramie (Boehmeria nivea L. Gaud) in response to PEG treatment, using illumina paired-end sequencing technology. Int. J. Mol. Sci. 2015, 16, 3493–3511. [Google Scholar] [CrossRef] [Green Version]
  29. Zotz, G.; Winkler, U. Aerial roots of epiphytic orchids: The velamen radicum and its role in water and nutrient uptake. Oecologia 2013, 171, 733–741. [Google Scholar] [CrossRef]
  30. Zou, L.H.; Wan, X.; Deng, H.; Zheng, B.Q.; Li, B.J.; Wang, Y. Data descriptor: RNA-seq transcriptomic profiling of crassulacean acid metabolism pathway in Dendrobium catenatum. Sci. Data 2018, 5, 180252. [Google Scholar] [CrossRef]
  31. Wang, A. Effects of bionic cultivation modes on yield and quality of Dendrobium officinale. J. Anhui Sci. Technol. Univ. 2022, 36, 44–48. (In Chinese) [Google Scholar] [CrossRef]
  32. Zhang, T.T.; Cui, Z.; Li, Y.X.; Kang, Y.Q.; Song, X.Q.; Wang, J.; Zhou, Y. Genome-wide identification and expression analysis of MYB transcription factor superfamily in Dendrobium catenatum. Front. Genet. 2021, 26, 714696. [Google Scholar] [CrossRef] [PubMed]
  33. Zhang, T.T.; Xu, Y.; Ding, Y.D.; Yu, W.G.; Wang, J.; Lai, H.G.; Zhou, Y. Identification and expression analysis of WRKY gene family in response to abiotic stress in Dendrobium catenatum. Front. Genet. 2022, 3, 800019. [Google Scholar] [CrossRef] [PubMed]
  34. Li, Y.X.; Zhang, T.T.; Xing, W.T.; Wang, J.; Yu, W.G.; Zhou, Y. Comprehensive genomic characterization of the NAC transcription factors and their response to drought stress in Dendrobium catenatum. Agronomy 2022, 12, 2753. [Google Scholar] [CrossRef]
  35. Zhang, G.Q.; Xu, Q.; Bian, C.; Tsai, W.C.; Yeh, C.M.; Liu, K.W.; Yoshida, K.; Zhang, L.S.; Chang, S.B.; Chen, F.; et al. The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution. Sci. Rep. 2016, 6, 19029. [Google Scholar] [CrossRef] [Green Version]
  36. Salih, H.; Gong, W.; He, S.; Sun, G.; Sun, J.; Du, X. Genome-wide characterization and expression analysis of MYB transcription factors in Gossypium hirsutum. BMC Genet. 2016, 17, 129. [Google Scholar] [CrossRef] [Green Version]
  37. Liu, Y.; Chai, M.; Zhang, M.; He, Q.; Su, Z.; Priyadarshani, S.V.G.N.; Liu, L.; Dong, G.; Qin, Y. Genome-wide analysis, characterization, and expression profile of the basic leucine zipper transcription factor family in pineapple. Int. J. Genom. 2020, 2020, 3165958. [Google Scholar] [CrossRef]
  38. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  39. Bray, N.L.; Pimentel, H.; Melsted, P.; Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 2016, 34, 525–527. [Google Scholar] [CrossRef]
  40. Wan, X.; Zou, L.H.; Zheng, B.Q.; Tian, Y.Q.; Wang, Y. Transcriptomic profiling for prolonged drought in Dendrobium catenatum. Sci. Data 2018, 5, 180233. [Google Scholar] [CrossRef]
  41. Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N.S.; Wang, J.T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003, 13, 2498–2504. [Google Scholar] [CrossRef] [PubMed]
  42. Seifert, E. OriginPro 9.1: Scientific data analysis and graphing software-software review. J. Chem. Inf. Model 2014, 54, 1552. [Google Scholar] [CrossRef]
  43. Szklarczyk, D.; Gable, A.L.; Lyon, D.; Junge, A.; Wyder, S.; Huerta-Cepas, J.; Simonovic, M.; Doncheva, N.T.; Morris, J.H.; Bork, P.; et al. STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019, 47, D607–D613. [Google Scholar] [CrossRef] [Green Version]
  44. Nijhawan, A.; Jain, M.; Tyagi, A.K.; Khurana, J.P. Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice. Plant Physiol. 2008, 146, 333–350. [Google Scholar] [CrossRef] [Green Version]
  45. Malik, W.A.; Afzal, M.; Chen, X.; Cui, R.; Lu, X.; Wang, S.; Wang, J.; Mahmood, I.; Ye, W. Systematic analysis and comparison of ABC proteins superfamily confer structural, functional and evolutionary insights into four cotton species. Ind. Crop. Prod. 2021, 177, 114433. [Google Scholar] [CrossRef]
  46. Chuang, C.F.; Running, M.P.; Williams, R.W.; Meyerowitz, E.M. The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana. Genes Dev. 1999, 13, 334–344. [Google Scholar] [CrossRef] [Green Version]
  47. Hörberg, J.; Reymer, A. Specifically bound BZIP transcription factors modulate DNA supercoiling transitions. Sci. Rep. 2020, 10, 18795. [Google Scholar] [CrossRef]
  48. Zhao, P.; Ye, M.; Wang, R.; Wang, D.; Chen, Q. Systematic identification and functional analysis of potato (Solanum tuberosum L.) bZIP transcription factors and overexpression of potato bZIP transcription factor StbZIP-65 enhances salt tolerance. Int. J. Biol. Macromol. 2020, 15, 155–167. [Google Scholar] [CrossRef]
  49. Wang, J.; Zhou, J.; Zhang, B.; Vanitha, J.; Ramachandran, S.; Jiang, S.Y. Genome-wide expansion and expression divergence of the basic leucine zipper transcription factors in higher plants with an emphasis on sorghum. J. Integr. Plant Biol. 2011, 53, 212–231. [Google Scholar] [CrossRef]
  50. Pourabed, E.; Golmohamadi, F.G.; Monfared, P.S.; Razavi, S.M.; Shobbar, Z.S. Basic leucine zipper family in barley: Genome-wide characterization of members and expression analysis. Mol. Biotechnol. 2015, 57, 12–26. [Google Scholar] [CrossRef]
  51. Wei, K.; Chen, J.; Wang, Y.; Chen, Y.; Chen, S.; Lin, Y.; Pan, S.; Zhong, X.; Xie, D. Genome-wide analysis of bZIP-encoding genes in maize. DNA Res. 2012, 19, 463–476. [Google Scholar] [CrossRef] [Green Version]
  52. Zhou, Y.; Xu, D.; Jia, L.; Huang, X.; Ma, G.; Wang, S.; Zhu, M.; Zhang, A.; Guan, M.; Lu, K.; et al. Genome-wide identification and structural analysis of bZIP transcription factor genes in Brassica napus. Genes 2017, 8, 288. [Google Scholar] [CrossRef] [Green Version]
  53. Jiang, M.; Wang, Z.; Ren, W.; Yan, S.; Xing, N.; Zhang, Z.; Li, H.; Ma, W. Identification of the bZIP gene family and regulation of metabolites under salt stress in Isatis indigotica. Front. Plant Sci. 2022, 13, 1011616. [Google Scholar] [CrossRef] [PubMed]
  54. Manzoor, M.A.; Manzoor, M.M.; Li, G.; Abdullah, M.; Han, W.; Wenlong, H.; Shakoor, A.; Riaz, M.W.; Rehman, S.; Cai, Y. Genome-wide identification and characterization of bZIP transcription factors and their expression profile under abiotic stresses in Chinese pear (Pyrus bretschneideri). BMC Plant Biol. 2021, 21, 413. [Google Scholar] [CrossRef]
  55. Yang, Y.; Li, J.; Li, H.; Yang, Y.; Guang, Y.; Zhou, Y. The bZIP gene family in watermelon: Genome-wide identification and expression analysis under cold stress and root-knot nematode infection. PeerJ 2019, 7, e7878. [Google Scholar] [CrossRef] [Green Version]
  56. Wang, W.; Wang, Y.; Zhang, S.; Xie, K.; Zhang, C.; Xi, Y.; Sun, F. Genome-wide analysis of the abiotic stress-related bZIP family in switchgrass. Mol. Biol. Rep. 2020, 47, 4439–4454. [Google Scholar] [CrossRef]
  57. Zhang, W.; Ye, S.; Du, Y.; Zhao, Q.; Du, J.; Zhang, Q. Identification and expression analysis of bZIP members under abiotic stress in mung bean (Vigna radiata). Life 2022, 12, 938. [Google Scholar] [CrossRef]
  58. Choi, H.; Hong, J.; Ha, J.; Kang, J.; Kim, S.Y. ABFs, a family of ABA-responsive element binding factors. J. Biol. Chem. 2000, 275, 1723–1730. [Google Scholar] [CrossRef] [Green Version]
  59. Finkelstein, R.R.; Lynch, T.J. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 2000, 12, 599–609. [Google Scholar] [CrossRef] [Green Version]
  60. Lopez-Molina, L.; Mongrand, S.; Chua, N.H. A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis. Proc. Natl. Acad. Sci. USA 2001, 98, 4782–4787. [Google Scholar] [CrossRef]
  61. Wellmer, F.; Schäfer, E.; Harter, K. The DNA binding properties of the parsley bZIP transcription factor CPRF4a are regulated by light. J. Biol. Chem. 2001, 276, 6274–6279. [Google Scholar] [CrossRef] [Green Version]
  62. Chattopadhyay, S.; Ang, L.H.; Puente, P.; Deng, X.W.; Wei, N. Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. Plant Cell 1998, 10, 673–683. [Google Scholar] [CrossRef] [Green Version]
  63. Zhang, Y.; Gao, W.; Li, H.; Wang, Y.; Li, D.; Xue, C.; Liu, Z.; Liu, M.; Zhao, J. Genome-wide analysis of the bZIP gene family in Chinese jujube (Ziziphus jujuba Mill.). BMC Genom. 2020, 21, 483. [Google Scholar] [CrossRef]
  64. Zhou, Y.; Zeng, L.; Chen, R.; Wang, Y.; Song, J. Genome-wide identification and characterization of stress-associated protein (SAP) gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula. Arch. Biol. Sci. 2018, 70, 87–98. [Google Scholar] [CrossRef]
  65. Hu, W.; Wang, L.; Tie, W.; Yan, Y.; Ding, Z.; Liu, J.; Li, M.; Peng, M.; Xu, B.; Jin, Z. Genome-wide analyses of the bZIP family reveal their involvement in the development, ripening and abiotic stress response in banana. Sci. Rep. 2016, 22, 30203. [Google Scholar] [CrossRef] [Green Version]
  66. Jin, Z.; Xu, W.; Liu, A. Genomic surveys and expression analysis of bZIP gene family in castor bean (Ricinus communis L.). Planta 2014, 239, 299–312. [Google Scholar] [CrossRef]
  67. Sornaraj, P.; Luang, S.; Lopato, S.; Hrmova, M. Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: A molecular model of a wheat bZIP factor and implications of its structure in function. Biochim. Biophys. Acta 2016, 1860, 46–56. [Google Scholar] [CrossRef] [PubMed]
  68. Fujita, Y.; Yoshida, T.; Yamaguchi-Shinozaki, K. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiol. Plant 2013, 147, 15–27. [Google Scholar] [CrossRef]
  69. Liu, J.X.; Srivastava, R.; Che, P.; Howell, S.H. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. Plant J. 2007, 51, 897–909. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Zhang, Z.; Chen, X.; Guan, X.; Liu, Y.; Chen, H.; Wang, T.; Mouekouba, L.D.; Li, J.; Wang, A. A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members’ expression in tomato. Biosci. Biotechnol. Biochem. 2014, 78, 1337–1349. [Google Scholar] [CrossRef]
  71. Wong, M.M.; Bhaskara, G.B.; Wen, T.N.; Lin, W.D.; Nguyen, T.T.; Chong, G.L.; Verslues, P.E. Phosphoproteomics of Arabidopsis highly ABA-induced 1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation. Proc. Natl. Acad. Sci. USA 2019, 116, 2354–2363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Danquah, A.; de Zelicourt, A.; Colcombet, J.; Hirt, H. The role of ABA and MAPK signaling pathways in plant abiotic stress responses. Biotechnol. Adv. 2014, 32, 40–52. [Google Scholar] [CrossRef] [PubMed]
  73. Liu, S.Y.; Zhang, C.B.; Zhu, Q.W.; Guo, F.; Chai, R.; Wang, M.X.; Deng, X.Y.; Dong, T.T.; Meng, X.Q.; Zhu, M.K. Genome-and transcriptome-wide systematic characterization of bZIP transcription factor family identifies promising members involved in abiotic stress response in sweetpotato. Sci. Hortic. 2022, 303, 111185. [Google Scholar] [CrossRef]
  74. Sun, X.; Li, Y.; Cai, H.; Bai, X.; Ji, W.; Ding, X.; Zhu, Y. The Arabidopsis AtbZIP1 transcription factor is a positive regulator of plant tolerance to salt, osmotic and drought stresses. J. Plant. Res. 2012, 125, 429–438. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Phylogenetic analyses of bZIP proteins from D. catenatum, Arabidopsis and rice. A phylogenetic tree of bZIP proteins was constructed using MEGA 6.0 software. The 10 subgroups are indicated by different colors and different colored letters. The blue circles represent A. thaliana bZIPs (AtbZIPs), the red stars represent D. catenatum bZIPs (DcbZIPs) and the black triangles represent O. sativa bZIPs (OsbZIPs).
Figure 1. Phylogenetic analyses of bZIP proteins from D. catenatum, Arabidopsis and rice. A phylogenetic tree of bZIP proteins was constructed using MEGA 6.0 software. The 10 subgroups are indicated by different colors and different colored letters. The blue circles represent A. thaliana bZIPs (AtbZIPs), the red stars represent D. catenatum bZIPs (DcbZIPs) and the black triangles represent O. sativa bZIPs (OsbZIPs).
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Figure 2. Motifs, structures and phylogenetic relationships of DcbZIP family members. (A) A phylogenetic tree of 62 DcbZIP proteins constructed using the maximum likelihood method. The different subgroups are indicated by different background colors and letters. (B) Conserved motifs of DcbZIP proteins. Different motifs are represented by different colored boxes. (C) Exon/intron structures of DcbZIP genes with UTR(s), exon(s) and intron(s) indicated with green boxes, yellow boxes and black lines, respectively. The phylogenetic tree, conserved motifs and gene structures were predicted with TBtools.
Figure 2. Motifs, structures and phylogenetic relationships of DcbZIP family members. (A) A phylogenetic tree of 62 DcbZIP proteins constructed using the maximum likelihood method. The different subgroups are indicated by different background colors and letters. (B) Conserved motifs of DcbZIP proteins. Different motifs are represented by different colored boxes. (C) Exon/intron structures of DcbZIP genes with UTR(s), exon(s) and intron(s) indicated with green boxes, yellow boxes and black lines, respectively. The phylogenetic tree, conserved motifs and gene structures were predicted with TBtools.
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Figure 3. Analysis of cis-elements in the DcbZIP genes promoter regions. (A) A phylogenetic tree of 62 DcbZIP proteins was constructed with the ML method. The different subgroups are indicated by different background colors and letters. (B) The different colored blocks represent the different types of cis-elements and their locations in each DcbZIP gene. (C) The different colors and numbers in the grid indicate the numbers of different promoter elements in the DcbZIP genes. The types, numbers and locations of potential elements in the promoter regions 2-kb upstream of the DcbZIP genes were analyzed by PlantCARE.
Figure 3. Analysis of cis-elements in the DcbZIP genes promoter regions. (A) A phylogenetic tree of 62 DcbZIP proteins was constructed with the ML method. The different subgroups are indicated by different background colors and letters. (B) The different colored blocks represent the different types of cis-elements and their locations in each DcbZIP gene. (C) The different colors and numbers in the grid indicate the numbers of different promoter elements in the DcbZIP genes. The types, numbers and locations of potential elements in the promoter regions 2-kb upstream of the DcbZIP genes were analyzed by PlantCARE.
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Figure 4. Hierarchical clustering of DcbZIP genes expression profiles across different D. catenatum tissues. Data were normalized relative to each gene’s mean expression value across all tissues and log2−transformed. TPM (transcripts per million) values were used to create heat maps showing the expressions of DcbZIP genes in different tissues. The expression level ranges from low expression (green) to high expression (red).
Figure 4. Hierarchical clustering of DcbZIP genes expression profiles across different D. catenatum tissues. Data were normalized relative to each gene’s mean expression value across all tissues and log2−transformed. TPM (transcripts per million) values were used to create heat maps showing the expressions of DcbZIP genes in different tissues. The expression level ranges from low expression (green) to high expression (red).
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Figure 5. The expression patterns of DcbZIP genes evaluated by RNA-seq. The left heatmap shows the expressions of DcbZIP genes under different volumetric water contents of base material. Data were normalized relative to each gene’s mean expression value across all treatments and log2-transformed. TPM (transcripts per million) values were used to create heat map showing the expression of DcbZIP genes. The expression levels range from low expression (green) to high expression (red). The right heatmap shows the TPM ratios, with high ratios in light orange and low ratios in cyan. T1 was equal to the TPM of 10–15% divided by the TPM of 30–35%, and T2 was equal to the TPM of 0% divided by the TPM of 30–35%. Ranges of 30–35%, 10–15% and 0% represent the volumetric water content of the base material declining to ~30–35%, ~10–15% and ~0%, respectively.
Figure 5. The expression patterns of DcbZIP genes evaluated by RNA-seq. The left heatmap shows the expressions of DcbZIP genes under different volumetric water contents of base material. Data were normalized relative to each gene’s mean expression value across all treatments and log2-transformed. TPM (transcripts per million) values were used to create heat map showing the expression of DcbZIP genes. The expression levels range from low expression (green) to high expression (red). The right heatmap shows the TPM ratios, with high ratios in light orange and low ratios in cyan. T1 was equal to the TPM of 10–15% divided by the TPM of 30–35%, and T2 was equal to the TPM of 0% divided by the TPM of 30–35%. Ranges of 30–35%, 10–15% and 0% represent the volumetric water content of the base material declining to ~30–35%, ~10–15% and ~0%, respectively.
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Figure 6. Comprehensive analysis of drought-related DcbZIP genes. (A) Correlation analysis of 33 upregulated DcbZIP genes. Red indicates a positive correlation and blue indicates a negative correlation. The circle size indicates the absolute value of the correlation coefficient. (B) Co-expression network analysis. The red lines represent a positive correlation. The size represents the degree calculated by the Cytoscape. The four polygons situated in the center represent core genes with high connectivity.
Figure 6. Comprehensive analysis of drought-related DcbZIP genes. (A) Correlation analysis of 33 upregulated DcbZIP genes. Red indicates a positive correlation and blue indicates a negative correlation. The circle size indicates the absolute value of the correlation coefficient. (B) Co-expression network analysis. The red lines represent a positive correlation. The size represents the degree calculated by the Cytoscape. The four polygons situated in the center represent core genes with high connectivity.
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Figure 7. Interaction networks of the DcbZIPs in D. catenatum and their orthologs in Arabidopsis. The network was predicted by STRING. The DcbZIP proteins were shown in the red font below with the Arabidopsis orthologs in the red dashed box. Empty nodes represent proteins with unknown 3D structures. Filled nodes represent known or predicted 3D structures. Light blue lines and rose lines represent known interactions. Green, orange and blue lines represent predicted interactions. Cyan lines represent text mining, black lines represent co-expression and light purple lines represent protein homology.
Figure 7. Interaction networks of the DcbZIPs in D. catenatum and their orthologs in Arabidopsis. The network was predicted by STRING. The DcbZIP proteins were shown in the red font below with the Arabidopsis orthologs in the red dashed box. Empty nodes represent proteins with unknown 3D structures. Filled nodes represent known or predicted 3D structures. Light blue lines and rose lines represent known interactions. Green, orange and blue lines represent predicted interactions. Cyan lines represent text mining, black lines represent co-expression and light purple lines represent protein homology.
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Figure 8. Real-time quantitative PCR analyses of the 17 core DcbZIP genes under drought stress in roots, stems and leaves. The mean expression value was calculated from three replicates. Vertical bars indicate the standard deviation. Values of 0, 3, 6, 9, 12, 24 and 48 indicate hours after treatment. Mean values and standard deviations were calculated according to the data. The unstressed level (0 h) was used as a control. Asterisks (* or **) indicate a significant difference at p < 0.05 or p < 0.01, respectively.
Figure 8. Real-time quantitative PCR analyses of the 17 core DcbZIP genes under drought stress in roots, stems and leaves. The mean expression value was calculated from three replicates. Vertical bars indicate the standard deviation. Values of 0, 3, 6, 9, 12, 24 and 48 indicate hours after treatment. Mean values and standard deviations were calculated according to the data. The unstressed level (0 h) was used as a control. Asterisks (* or **) indicate a significant difference at p < 0.05 or p < 0.01, respectively.
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Table
Table 1. Physicochemical properties of D. catenatum bZIP proteins.
Table 1. Physicochemical properties of D. catenatum bZIP proteins.
Gene NameGene IDLocusLengthMW(kDa)pISubcellular LocalizationGroup
DcbZIP1LOC110102063NW_021318745.1:471271…48657215316.979.98nuclearH
DcbZIP2LOC110094784NW_021319121.1:1711930…171337825428.366.6nuclearE
DcbZIP3LOC110115573NW_021320155.1:215…146215517.669.03nuclearS
DcbZIP4LOC110112903NW_021359921.1: 258…81613815.5911.53nuclearS
DcbZIP5LOC110095238NW_021319911.1:476590…47701214015.6711.58nuclearS
DcbZIP6LOC110113945NW_021318751.1:434550…43535114616.7610.47nuclearS
DcbZIP7LOC110114248NW_021318952.1:1141827…114296614917.1611.09nuclearS
DcbZIP8LOC110109372NW_021318890.1:45595…4669013015.248.49nuclearS
DcbZIP9LOC110095100NW_021319641.1:962641…96671235638.765.18nuclearC
DcbZIP10LOC110099409NW_021319484.1:1588342…160486431134.375.57nuclearC
DcbZIP11LOC110116565NW_021394673.1:450792…45457631735.956.49mitochondrialS
DcbZIP12LOC110098756NW_021319202.1:1038678…104647033838.289.30nuclearA
DcbZIP13LOC110100055NW_021319455.1:997836…100067522224.825.91nuclearA
DcbZIP14LOC110112185NW_021318640.1:185732…18705326528.719.30mitochondrialA
DcbZIP15LOC110115326NW_021319048.1:847890…87225140642.699.13cytoplasmicA
DcbZIP16LOC110096541NW_021319431.1:31825…5018740943.336.33nuclearG
DcbZIP17LOC110099255NW_021319682.1:13603178…1360786368374.166.38nuclearB
DcbZIP18LOC110104466NW_021319842.1:67813…11127039843.506.16nuclearI
DcbZIP19LOC110116528NW_021319455.1:2008465…201239025928.906.41nuclearE
DcbZIP20LOC110113515NW_021320183.1:270501…30171846651.396.36nuclearD
DcbZIP21LOC110096667NW_021319983.1:403141…41256448954.635.71nuclearD
DcbZIP22LOC110113543NW_021320019.1:9020891…902208716718.718.66nuclearS
DcbZIP23LOC110102116NW_021318705.1:1911115…192423427329.495.94nuclearD
DcbZIP24LOC110110500NW_021319360.1:1021557…102360932136.955.29nuclearF
DcbZIP25LOC110109220NW_021319982.1:676693…69019636239.535.89nuclearC
DcbZIP26LOC110091873NW_021319588.1:431917…45012442747.226.82nuclearD
DcbZIP27LOC110102106NW_021318705.1:1988364…198973516218.009.76nuclearA
DcbZIP28LOC110096161NW_021318623.1:23442…2778469274.946.08nuclearB
DcbZIP29LOC110116680NW_021319255.1:1051643…105668654259.416.21nuclearI
DcbZIP30LOC110099156NW_021318726.1:1712729…171911852256.716.21nuclearI
DcbZIP31LOC110102661NW_021319579.1:559884…58321435038.196.88nuclearI
DcbZIP32LOC110106152NW_021318595.1:5943723…594693922625.6511.36nuclearS
DcbZIP33LOC110096611NW_021319126.1:383344…39814533937.156.13nuclearI
DcbZIP34LOC110103109NW_021320146.1:675175…67882731934.655.90nuclearE
DcbZIP35LOC110098225NW_021318839.1:350918…36787131134.839.50nuclearA
DcbZIP36LOC110092531NW_021318711.1:1796647…180632839642.689.19nuclearA
DcbZIP37LOC110109339NW_021318998.1:251776…26029640743.507.70nuclearA
DcbZIP38LOC110097322NW_021319772.1:217773…21988414115.697.65nuclearS
DcbZIP39LOC110114883NW_021318595.1:12862132…1286468435738.967.60nuclearA
DcbZIP40LOC110107968NW_021319536.1:408463…44508427730.195.19nuclearA
DcbZIP41LOC110095320NW_021318963.1:1120306…113017235938.077.11nuclearG
DcbZIP42LOC110094070NW_021318618.1:1794449…179505415918.317.07nuclearS
DcbZIP43LOC110109278NW_021416702.1:86653…8739518321.416.72nuclearS
DcbZIP44LOC110099470NW_021318619.1:750999…75192413215.6510.09nuclearS
DcbZIP45LOC110110747NW_021318796.1:2352740…237058142747.268.46nuclearD
DcbZIP46LOC110094737NW_021319121.1:686641…69556336540.637.06nuclearD
DcbZIP47LOC110110388NW_021319315.1:2104944…212560238142.917.68nuclearD
DcbZIP48LOC110094083NW_021318700.1:245031…24664314616.348.71nuclearH
DcbZIP49LOC110091793NW_021318565.1:363970…37084444750.017.32nuclearD
DcbZIP50LOC110112805NW_021318937.1:66248…7325943348.477.75nuclearD
DcbZIP51LOC110105751NW_021318677.1:122368…13535634137.246.22nuclearI
DcbZIP52LOC110114853NW_021319083.1:8027963…805044339442.695.44nuclearI
DcbZIP53LOC110099371NW_021319442.1:514414…51559114316.698.85nuclearS
DcbZIP54LOC110109693NW_021378092.1:460796…46911538641.585.14nuclearG
DcbZIP55LOC110094315NW_021319161.1:846164…85831526930.978.75nuclearG
DcbZIP56LOC110101858NW_021319436.1:2509122…251908516118.079.44nuclearH
DcbZIP57LOC110111774NW_021318920.1:351221…35873536941.595.68nuclearD
DcbZIP58LOC110100891NW_021318787.1:716526…71741013015.248.49nuclearS
DcbZIP59LOC110095296NW_021319910.1:646885…65565140144.125.79nuclearI
DcbZIP60LOC110113665NW_021324968.1:31584…3439833537.955.13nuclearB
DcbZIP61LOC110101931NW_021319436.1:1660963…167753136139.458.47nuclearI
DcbZIP62LOC110106828NW_021319013.1:318529…32028927329.159.73nuclearA
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Wang, P.; Li, Y.; Zhang, T.; Kang, Y.; Li, W.; Wang, J.; Yu, W.; Zhou, Y. Identification of the bZIP Gene Family and Investigation of Their Response to Drought Stress in Dendrobium catenatum. Agronomy 2023, 13, 236. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13010236

AMA Style

Wang P, Li Y, Zhang T, Kang Y, Li W, Wang J, Yu W, Zhou Y. Identification of the bZIP Gene Family and Investigation of Their Response to Drought Stress in Dendrobium catenatum. Agronomy. 2023; 13(1):236. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13010236

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Wang, Peng, Yuxin Li, Tingting Zhang, Yuqian Kang, Wei Li, Jian Wang, Wengang Yu, and Yang Zhou. 2023. "Identification of the bZIP Gene Family and Investigation of Their Response to Drought Stress in Dendrobium catenatum" Agronomy 13, no. 1: 236. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13010236

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