Comprehensive Analysis of the Poplar DREB A4 Subfamily and the Role of PtrDREB4 in Response to Drought Stress

TL;DR

This study explores the DREB A4 gene subfamily in poplar and shows that one gene, PtrDREB4, helps plants tolerate drought stress.

Contribution

The study identifies and functionally characterizes the DREB A4 subfamily in poplar, revealing the role of PtrDREB4 in drought tolerance.

Findings

29 DREB A4 genes were identified in the poplar genome, with 19 undergoing segmental duplication.

PtrDREB4 overexpression in Arabidopsis and yeast enhanced drought tolerance.

DREB A4 genes showed distinct expression patterns and stress-related promoter elements.

Abstract

The dehydration response element binding protein (DREB) family of the AP2/ERF superfamily functions as a key regulatory component in plant adaptation to water-deficit conditions. However, studies on the DREB A4 subfamily in poplar (Populus trichocarpa) are insufficient. In this study, members of the DREB A4 subgroup in poplar were identified and analyzed via bioinformatic analysis. A pCAMBIA-2300-PtrDREB4 expression vector was constructed and transformed into Arabidopsis, followed by phenotypic analysis of transgenic plant in response to drought stress. A total number of 29 DREB A4 members were identified in the poplar genome. Synteny analysis revealed that 19 gene pairs went through segmental duplication at least 12.84 million years ago. Their promoter regions were enriched with cis-elements related to stress resistance, hormone regulation, and growth and development. Upstream regulator analysis of poplar DREB A4 genes identified 425 transcription factor genes, which belonged to 39 families. Gene expression analysis demonstrated distinct expression patterns of DREB A4 genes in leaves, roots and stems with a notable response to drought stress. Ectopic expression of PtrDREB4 in yeast and Arabidopsis increased the drought tolerance of transformants, indicating the positive role of PtrDREB4 in response to drought stress. These findings collectively established a theoretical foundation for further functional exploration of DREB A4 genes in poplar.

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1. Introduction

Abiotic stresses, notably drought, significantly constrain agricultural productivity [1]. To enable survival under abiotic stress, plants employ an array of physiological and molecular adaptations [2]. Among these, transcription factors (TFs) play a pivotal role by orchestrating the expression of downstream genes [3]. In particular, DREBs, a type of plant-specific family of TFs, play a pivotal role in mediating plant responses to drought and other abiotic stresses.

The DREB family belongs to the AP2/ERF (APETALA2/ethylene responsive element-binding factor) superfamily, and can specifically bind to DRE/CRT (G/ACCGAC) cis-elements to cope with abiotic stress. These proteins typically contain a single AP2 domain of 60–70 amino acids, comprising two conserved blocks, YRG and RAYD elements [4]. The main function of YRG element is to bind to the cis-acting elements of target genes. The amino acids V14 and E19 within AP2 domain are crucial for this binding, with V14 being particularly critical for DNA-binding specificity. RAYD element mainly regulated the binding of AP2 conserved domains to cis-acting elements through structural conformation or protein–protein interactions [4]. Based on sequence features flanking the AP2 domain, DREB members are categorized into six subfamilies: A1-A6 [5].

DREB genes have been identified across a range of plant species, including Arabidopsis, Moso bamboo, foxtail millet, tobacco, potato, wild soybean, and poplar [6,7,8,9,10,11]. However, most studies have focused on the A1 and A2 subfamilies, with limited research on the members in DREB A4 subfamily. Nevertheless, some functional studies have been conducted. For example, the heterologous expression of Eucalyptus DREB A4 member EgrDREB3 in Arabidopsis conferred tolerance to low temperature and salinity, and delayed leaf senescence through JA-dependent and JA-independent pathways [12]. In tomato, SlDREBA4 was upregulated under high ABA, cold, drought and high-salt conditions [13]. Similarly, Arabidopsis HARDY and TINY—both DREB A4 members—improve water-use efficiency or drought resistance [14,15]. In Arabidopsis, transgenic plants expressing TINY exhibited stunted growth and promoted drought resistance with activated drought-responsive gene expression [16]. In poplar, seventy-five DREB genes have been identified, with PtrDREB17, PtrDREB18 and PtrDREB32 from the DREB A6 and A5 subfamilies showing significant upregulation under cold treatment [11]. The PeDREB2 gene from the A2 subfamily of the DREB family in Populus euphratica is involved in the plant’s response to salt stress [17]. Additional research has shown that PeDREB2L was predominantly expressed in flowers under dehydration, salt and ABA treatment [18]. Furthermore, heterologous expression of PeDREB2a in Arabidopsis confers enhanced tolerance to both drought and NaCl stress [19]. Moreover, poplar plants overexpressing DREB46 gene from the DREB A4 subfamily exhibited significantly improved drought tolerance [20].

Populus trichocarpa, a rapid-growing tree species with high ecological and economic value, is particularly susceptible to drought stress [21,22,23]. Despite the recognized importance of the DREB A4 subfamily in abiotic stress responses, its functional roles in woody plants have been limitedly described. In this study, we conducted a comprehensive investigation of the DREB A4 genes in poplar, focusing on their phylogenetic relationship, gene synteny, structural feature and expression pattern. Furthermore, we explored the specific role of PtrDREB4 in drought stress tolerance, with the aim of providing valuable insights for enhancing drought resistance in tree species.

2. Results

2.1. Twenty-Nine DREB A4 Subfamily Genes Are Identified in Poplar

To identify the potential candidate DREB A4 subfamily members in poplar, we utilized the AP2 domain (PF00847) to search the poplar database with HMMER. Meanwhile, 17 Arabidopsis DREB A4 proteins were employed to interrogate the poplar database with BLASTP. The candidate DREB A4 sequences were then confirmed through analysis with the CDD and SMART databases. As a result, 29 sequences were identified as DREB A4 gene family members in poplar, and named from PtrDREB1 to PtrDREB29 according to their chromosomal order (Table S2). The predicted protein lengths ranged from 175 (PtrDREB28) to 293 amino acids (PtrDREB26), with MW ranging from 18.84 kDa (PtrDREB13) to 31.56 kDa (PtrDREB26) and predicted pI from 4.77 (PtrDREB24) to 7.65 (PtrDREB7), respectively. Except PtrDREB7 (pI > 7), most of them were acidic proteins. Subcellular localization prediction demonstrated that 16 of these proteins were localized to the nucleus, one was found in the cytoplasm, and 12 were predicted to be distributed in both the nucleus and the cytoplasm (Table S2).

2.2. Segmental and Tandem Duplication Events Are Detected in Poplar DREB A4 Genes

We mapped the poplar DREB A4 family genes to poplar chromosomes using genome annotation. As shown in Figure 1, they were scattered and unevenly distributed on chromosomes 1, 2, 3, 6, 12, 13, 14, 15, 16, 18 and 19. Chromosome 3 contained the highest number, harboring five DREB A4 genes, while chromosome 12, 15 and 16 had only one of them.

A total of 19 pairs of segmental duplications and three pairs of tandem duplications were detected (Figure 1, Table S3). Based on Ks value analysis, the duplication events are dated to approximately 1.63 to 174.24 million years ago (MYA). In addition, the Ka/Ks ratio of all gene pairs were found to be less than one, suggesting that the duplicated genes have undergone purifying selection throughout their evolutionary history (Table S3).

We further studied the synteny relationships between poplar and Arabidopsis, and between poplar and rice. As shown in Figure 1, there were 24 and 10 homologous pairs, respectively. Four DREB A4 genes, PtrDREB2, PtrDREB5, PtrDREB12 and PtrDREB21 showed syntenic relationship with both Arabidopsis and rice genes, indicating these genes existed in ancestors before the divergence of monocots and dicots. Fifteen DREB A4 genes were found to have a many-to-one syntenic relationship with Arabidopsis and with rice. For example, PtrDREB1, PtrDREB13 and PtrDREB18 were associated with the same Arabidopsis gene (AT1G12630), and PtrDREB2, PtrDREB5 and PtrDREB12 were associated with the same rice gene (Os02g43940), suggesting these genes underwent lineage-specific expansion in poplar (Figure 1).

To infer the evolutionary history and predict the biological function of poplar DREB A4 genes, a phylogenetic tree containing 17 DREB A4 proteins from Arabidopsis, 12 proteins from rice and 29 proteins from poplar were generated (Figure 2). Based on the evolutionary tree, DREB A4 proteins showed the closer relationship between Arabidopsis and poplar (Figure 2).

2.3. Poplar DREB A4 Genes Show Highly Conserved Gene Structures, AP2 Domain Sequences and 3D Structures

Gene structures provide important clues for their functional diversification. The intron–exon organization of individual poplar DREB A4 genes were analyzed. DREB A4 genes contained 5′ UTR, 3′ UTR, 1 to 2 exons and 0 to 1 intron (Supplementary Figure S1A). In detail, one intron was found in PtrDREB9, while none was found in the coding sequence of other DREB A4 genes. The intron–exon structures of the DREB A4 genes showed a highly conserved pattern. Then, we analyzed their conserved domains. As expected, all of the DREB A4 proteins contained one AP2 domain (Figure S1B). Conserved motif analysis shed light on gene diversification. A total of 10 distinct motifs were found (Figure S1C). AP2 domain was jointly constituted of motif 1, 2 and 3, and all of the DREB A4 proteins contained motif 1, 2 and 3. Motif 1 belonged to RAYD element and motif 3 was part of the YRG element (Figure S1D).

Multiple sequence alignment revealed a high degree of conservation in AP2 domain among poplar DREB A4 proteins (Figure 3A). Apart from the characteristic YRG and RAYD motif, all DREB A4 proteins contained the same amino acid at positions 14 (V), and 24 proteins (PtrDREB1, PtrDREB3, PtrDREB5, PtrDREB6, PtrDREB7, PtrDREB8, PtrDREB10, PtrDREB11, PtrDREB13, PtrDREB14, PtrDREB15, PtrDREB16, PtrDREB17, PtrDREB18, PtrDREB19, PtrDREB20, PtrDREB21, PtrDREB22, PtrDREB23, PtrDREB24, PtrDREB25, PtrDREB26, PtrDREB28, and PtrDREB29) contained the same amino acid at position 19 (E) (Figure 3A). Furthermore, their AP2 domains consisted of three conserved β-sheets and an α-helix (Figure 3B,C and Figure S2).

2.4. Poplar DREB A4 Promoters Contain the Key Cis-Elements Related to Growth, Development, Phytohormone and Stress Response

To detect putative regulatory elements, we analyzed promoter sequences (2000 bp) of the DREB A4s using PlantCARE database 5.0. Multiple cis-acting elements mainly related to environmental stress, phytohormone, and growth and development were identified (Figure 4). The stress-related cis-acting elements included anaerobic induction (ARE), low-temperature responsiveness (LTR), MYB binding site involved in drought-inducibility (MBS), defense and stress responsiveness (TC-rich repeats) and wound-responsive elements (WUN-motif). In addition, various hormone-responsive elements, including ABA (ABRE), MeJA (CGTCA-motif and TGACG-motif), GA (GARE-motif, P-box), auxin (TGA-element) and SA (TCA-element) were also identified, implying the possible participation of poplar DREB A4 genes in various hormones signaling (Figure 4).

2.5. Poplar DREB A4 Genes Are Potentially Regulated by Multiple Transcription Factors

In order to predict TFs that may target poplar DREB A4, the promoter sequences were used as input in the Plant Transcriptional Regulatory Map (PTRM). A total of 425 TF genes, belonging to 39 TF families, were predicted to be the potential regulators of poplar DREB A4 genes (Figure 5A, Table S4). The most abundant TF family was ERF (263), followed by NAC (138), Dof (135), MYB (108) and C2H2 (70), while the least TF family was LFY (1), VOZ (1), FAR1 (1), C3H (1) and S1Fa-like (1) (Figure 5B, Table S4). Among the poplar DREB A4 genes, PtrDREB18 (79) and PtrDREB26 (79) had the most regulators (Table S4). Then, the top 10 highly enriched gene families, including B3, BBR-BPC, bZIP, C2H2, Dof, ERF, MIKC-MADS, MYB, NAC, and TCP were identified (Figure 5C). A total of 25, 19, 9, 18, 23, 18, 22, 23, 17 and 15 poplar DREB A4 genes were respectively regulated by B3, BBR-BPC, bZIP, C2H2, Dof, ERF, MIKC_MADS, MYB, NAC and TCP (Figure 5D). These families may participate in the regulation of poplar development.

2.6. Poplar DREB A4 Genes Have Overlapped but Distinct Expression Patterns

We analyzed their expression level in different organs, including the first fully expanded leaf, immature leaf, young leaf, root tip, root, stem internode and stem node (Figure 6). PtrDREB4, PtrDREB5, PtrDREB21, PtrDREB25 and PtrDREB26 showed relatively high, whereas PtrDREB1, PtrDREB3, PtrDREB10, PtrDREB11, PtrDREB13, PtrDREB14, PtrDREB18, PtrDREB19, PtrDREB20, PtrDREB23, PtrDREB24, PtrDREB27, PtrDREB28 and PtrDREB29 showed low expression level in the tested organs (Figure 6). Specifically, PtrDREB8, PtrDREB12 and PtrDREB22 showed relatively higher expression level in leaves and root tips. However, PtrDREB6 and PtrDREB7 showed no expression in roots. PtrDREB15 and PtrDREB16 had the closest homology relationship, and they showed similar expression patterns, implying that they may perform conserved functions in poplar (Figure 6).

To explore the possible roles of these genes, the transcriptional changes in DREB A4 genes under drought stress were further analyzed. After being treated with drought for 5 and 7 days, the expression of PtrDREB3, PtrDREB4, PtrDREB11, PtrDREB18, PtrDREB19, PtrDREB26, PtrDREB28 and PtrDREB29 was significantly up-regulated. Differently, the expression of PtrDREB5, PtrDREB6, PtrDREB12, PtrDREB13, PtrDREB14, PtrDREB15, PtrDREB17, PtrDREB21, PtrDREB23, PtrDREB24 and PtrDREB25 were significantly down-regulated. These findings imply that some DREB A4 genes were crucial in response to drought stress (Figure 7).

2.7. PtrDREB4 Positively Regulates Osmotic Tolerance in Transgenic Yeast

Given the dual patterns of broad tissue expression and drought-responsive induction observed for PtrDREB4, we constructed transformed yeast lines to test its role in drought stress adaptation (Figure 6 and Figure 7). pYES2-PtrDREB4 and pYES2 empty vector (control) were respectively introduced in to the yeast cells. We observed that the growth rate of both transformant cells was similar under normal condition (Figure 8A). But, in the presence of a high concentration of mannitol, PtrDREB4-transformed yeast survived much better than the control did (Figure 8A). Therefore, PtrDREB4 expression increased tolerance to osmotic stress in yeast cells.

2.8. PtrDREB4 Increases the Drought Tolerance of Transgenic Plants

To further investigate whether the expression of PtrDREB4 enhances drought tolerance in transgenic plants, we obtained eight independent transgenic lines. And, they all showed high expression of PtrDREB4 (Figure S3). From these, three independent lines (OE#6, OE#7 and OE#8) were selected for further analysis.

We first compared the germination rates of wild-type (WT) and transgenic plants under both normal and drought stress conditions. Statistical comparison revealed no significant difference in germination rates between WT and transgenic plants under normal conditions (Figure 8B,C). However, under drought stress treatment, the transgenic lines demonstrated notably higher germination rates compared to the WT (Figure 8B,C). Next, we evaluated the impact of PtrDREB4 expression on drought stress tolerance in transgenic Arabidopsis. We observed that while PtrDREB4 expression slowed the growth of transgenic plants, the transgenic lines exhibited reduced root lengths and fresh weights relative to the WT (Figure 9A,C,D). However, under treatment with 250 mM mannitol, transgenic lines exhibited less growth inhibition, as evidenced by longer primary roots and a lower growth inhibition rate compared to the WT (Figure 9B–E). These results suggest that PtrDREB4 expression confers improved osmotic stress tolerance in transgenic Arabidopsis.

3. Discussion

Poplar, a rapid-growing tree known for its substantial biomass accumulation in terrestrial ecosystem, held immense economic value due to its utilization in industries such as paper production, land reforestation and bioenergy feedstocks generation [24,25]. Notably, as a model tree species, its genome was successfully sequenced in 2006 [26]. The availability of this reference genome enabled the systematic identification of the DREB A4 gene family in poplar.

Our analysis revealed that the DREB A4 gene family in poplar comprises 29 members. The number was more than that which was reported previously (26) [11]. This variation in gene count may be attributed to our access to a more recent and complete genome assembly, as that of PtrDREB6 and PtrDREB8, PtrDREB10 and PtrDREB11, and PtrDREB15 and PtrDREB16 were respectively mapped to the same previously reported gene of PtrDREB52, PtrDREB47 and PtrDREB72. The number of DREB A4 genes are variable in different plant species. 2 in wild soybean, 16 in tomato, 17 in Arabidopsis, and 18 in potato were detected [6,9,10,27]. The variable numbers could be a result of different duplication progresses during plant evolution and different retention rates of genes after duplication [28]. Members of DREB A4 genes exhibited a clustered distribution across the 19 poplar chromosomes, a pattern that may have originated from localized genomic duplication (Figure 1). In addition, gene duplication serves as a key evolution force, fueling gene family expansion and fostering functional innovation and adaptive evolution in plants [29]. In this study, we verified that the expansion of poplar DREB A4 subfamily was primarily driven by segmental and tandem duplication events (Table S3). This is consistent with the situation in potato and tomato [9,27]. Ka/Ks ratio is routinely employed to estimate the selection intensity of duplicated genes and approximate dates of duplication events. Here, Ks values ranged from 0.03 to 3.17, alongside Ka/Ks ratios consistently under 1. The low Ka/Ks ratios indicated that the duplicated genes are subjected to purifying selection subsequent to the duplication events occurred between 1.63 and 174.24 MYA (Table S3). In terms of the structure, all the DREB A4 members showed highly conserved AP2 domain sequences and a 3D structure (Figure 3). Introns are important structural components of genes, and regulate alternative splicing and the evolution rate of genes. Intron insertion or deletion may affect gene function [30]. A previous study indicated that Arabidopsis DREB genes contained no intron [31]. Later studies revealed DREB genes in other species, such as potato, cotton and tomato, contained intron [9,27,32]. Notably, 86.2% (25 out of 29) of the poplar DREB A4 subfamily genes lacked introns, indicating a relatively conserved gene structure (Figure S1). Investigation of the motifs within protein sequences can offer clues regarding the evolution of plants [33]. Our MEME analysis unveiled the presence of diverse motifs in DREB A4 protein sequences (Figure S1).

Promoters are the switches of genes located upstream of genes, which contain cis-elements. Upstream regulatory genes turn on or off the functional activity of downstream targets via interaction with cis-elements in promoters [34]. An examination revealed a spectrum of elements involved in growth regulation and stress adaptation, as well as phytohormone interaction in DREB A4 promoters (Figure 4). Together, our data attest to the pivotal importance of DREB A4 genes in shaping stress response and physiological process within the poplar, consistent with the established role of BrDERBs in wucai [35].

Understanding the expression patterns of DREB A4 genes helps elucidate their physiological roles. For example, the high leaf expression of PtrDREB12, together with the documented role of its ortholog ERF34 (AT2G44940) in leaf senescence, suggests its involvement in leaf senescence [36] (Figure 6). Given that poplar is a primary raw material for paper, pulping, furniture and other industrial applications, the high expression of several DREB A4 genes in the stems holds significance [37]. Notably, PtrDREB13 was prominently expressed in stems. Its Arabidopsis ortholog, AT1G12630, is a known regulator of secondary cell wall synthesis, suggesting that PtrDREB13 may be important for wood formation [38].

Drought stress has profound global impacts on plant development and productivity through generating both osmotic and oxidative stresses [36]. In cotton and wild soybean, DREB genes showed varied stress responses [10,32]. Consistent with this, a parallel expression trend was observed in our study. The universal expression changes in DREB A4 genes under drought implicate them in stress perception and signaling (Figure 7). This was further evidenced by the presence of multiple stress- and hormone-related cis-elements in their promoters. (Figure 4). In a previous study, poplar DREB46 (name as PtrDREB28 here) was induced by PEG-6000 [20]. PtrDREB28 was induced by drought after treatment for 5 and 7 days. PtrDREB51 (name as PtrDREB22 here) was up-regulated by PEG after treatment for 4 and 8 h. PtrDREB22 was induced by 5 days of drought stress [11].

Heterologous expression of MnDREB4A in tobacco was shown to provide broad-spectrum resistance against multiple abiotic stresses [39]. Heterologous expression of DREB A4 gene, CiDREB3, improved Arabidopsis drought resistance [40]. In this study, PtrDREB4 exhibited robust induction under drought conditions (Figure 7). Biological function analysis in yeast and Arabidopsis system indicated that expression of PtrDREB4 conferred osmotic tolerance on transgenic yeast and Arabidopsis (Figure 8 and Figure 9). Promoter cis-element analysis of PtrDREB4 revealed the presence of multiple stress- and hormone-related regulatory motifs, including ABRE (ABA-responsive element), P-box (gibberellin-responsive element), TCA-element (salicylic acid-responsive element), and ARE (anaerobic induction element). The co-occurrence of these distinct cis-elements suggests that PtrDREB4 expression is potentially regulated by diverse signaling pathways, enabling it to integrate multiple environmental and hormonal cues. ABRE is a well-characterized cis-acting element that mediates ABA-dependent gene expression during osmotic stress, playing a central role in drought responses [41]. The presence of ABRE in the PtrDREB4 promoter is consistent with its induction under drought conditions observed in our study (Figure 7), and suggesting that PtrDREB4 may function downstream of ABA signaling to enhance stress tolerance. Collectively, our data indicate that PtrDREB4 enhances drought tolerance in poplar, although further investigation needs be conducted to reveal the corresponding regulatory mechanism.

4. Materials and Methods

4.1. Detection of DREB A4 Members in Poplar

P. trichocarpa reference sequence (version 3.0) and Arabidopsis DREB A4 reference protein sequences were retrieved from Phytozome platform and TAIR database, respectively [42]. HMMER software (version 3.0) was used to detect the candidate DREB A4 proteins with Pfam profile of AP2 domain: PF00847 [43]. Arabidopsis DREB A4 proteins were employed as queries in a BLASTP (version 2.12.0) search targeting the poplar proteins. We merged the protein sequences from the two approaches and subsequently eliminated all redundancies. Verification of AP2 domain in the candidate DREB A4 members was performed via the NCBI CDD and SMART database [44]. The molecular weight (MW), predict isoelectric points (pI), and grand average of hydropathicity (GRAVY) for each protein were determined with ExPASy. TBtools software (version 2.423) was used to detect instability and aliphatic index [45]. Plant-PLoc server was used to calculate subcellular localizations [46].

4.2. Chromosome Location, Synteny, Phylogeny Reconstruction, Gene Structure, Conserved Motif Identification

Chromosome positions of the DREB A4 genes were acquired from poplar annotation information. Gene duplication events and genomic synteny were assessed with TBtools. The Ka/Ks (nonsynonymous/synonymous substitution rate) were assessed with the KaKs_calculator (version 2.0) software. Using the molecular clock formula T = Ks/2λ (with λ = 9.1 × 10^−9^), we estimated the timing of the duplication event [21]. Phylogenetic analysis was conducted with the DREB A4 proteins of poplar, rice and Arabidopsis using MEGA 11 [47]. Gene structure analysis was conducted with gene annotation information [26]. MEME was leveraged to delineate conserved motifs within DREB A4 proteins (maximum number = 10) [48].

4.3. Sequence Alignment and Three-Dimensional Structure Analysis

Sequence alignments between the AP2 domains of DREB A4 proteins were performed using Jalview version 2 software released in 2009 [49]. Sequence logos for the AP2 domain were performed at WebLogo (https://weblogo.threeplusone.com/). The three-dimensional (3D) architecture of DREB A4 were generated with SWISS-MODEL and visualized with Pymol 3.0.0 software.

4.4. Cis-Acting Element and TF Regulatory Network Analysis

Upstream 2 kb promoter of DREB A4s were analyzed for cis-acting elements via PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [50]. The upstream regulatory TFs were predicted via PTRM with upstream sequences (2 kb) of DREB A4 genes (p-value ≤ 1 × 10^−5^). Network visualization was performed with Cytoscape (version 3.10.4) [51]. The wordcloud was generated using R software with wordcloud2 package (version 4.3.3).

4.5. Plant Material and Stress Treatments

We used Populus davidiana × Populus bolleana plants in this study. Plants were grown in a greenhouse under a 16 h light/8 h dark photoperiod, with a temperature of 25 °C (day)/23 °C (night) and relative humidity maintained at 50–60%. To impose drought treatment, water was withheld from two-month-old plants with similar growth vigor for 0 day, 5 and 7 days, and the mature leaves were immediately stored at −80 °C for later use.

4.6. Expression Pattern Analysis of DREB A4 Genes

Transcriptome data were obtained from phytozome for the following poplar tissues: first fully expanded leaf, immature leaf, young leaf, root tip, root, stem internode and stem node. The heatmap was constructed in TBtools to display the log_2_FPKM values [44].

4.7. Quantitative RT-PCR (qRT-PCR) Analysis of Poplar DREB A4 Genes

Total RNA was isolated and subjected to reverse transcription following the manufacturer’s instruction of the kit (Vazyme Biotech CO., Ltd., Nanjing, China, R212-01). qRT-PCR analysis was conducted using the Bio-Red CFX96 system. Expression levels were quantified via the 2^−ΔΔCT^ method, and the primers used are listed in Table S1.

4.8. Stress Tolerance Assay in Transgenic Yeast

For heterologous expression in yeast, the PtrDREB4 coding sequence was subcloned into the pYES2 vector (Beijing Coolaber Science&Technology Co., Ltd., Beijing, China, Cat# YM4000). Then, resultant pYES2-PtrDREB4 plasmid was introduced into INVSc1 competent cells following the manufacturer’s instructions (Beijing Coolaber Science&Technology Co., Ltd., China, Cat# CC303). To analyze potential function of PtrDREB4, yeast transformants were first precultured overnight in SC-ura medium with 2% glucose. For induction, cells were harvested, normalized to an OD_600_ of 0.4 in fresh SC-ura medium containing 2% galactose, and cultured for 24 h. Cultures were then adjusted to an OD_600_ of 1.8. For the control, yeast cells were incubated in sterile water. For drought stress assays, yeast cells were cultured in 2 M mannitol for 48 h. Then, serial dilutions (1:1, 1:10, 1:100, 1:1000, 1:10,000) were prepared with the transformed yeast cells. For the spot assay, 3 μL of each yeast culture was plated onto SC-ura containing 2% glucose and grown at 30 °C for 48 h.

4.9. Arabidopsis Transformation and Stress Treatment

Coding sequence of PtrDREB4 was inserted into the pCAMBIA-2300 vector, with expression driven by a duplicated CaMV 35S promoter. The resulting plasmid was transferred into Arabidopsis thaliana (Col-0) via GV3101. T_3_ homozygous plants were identified for subsequent phenotypic characterization. To detect the germination rate, the medium was supplemented with 0 or 300 mM mannitol. To detect the primary root length and biomass, the medium was supplemented with 0 or 250 mM mannitol.

4.10. Statistical Analysis

Statistical analysis was performed using Microsoft Excel. Data were analyzed by two-tailed Student’s t-test, with significance defined as p < 0.05. All data were obtained from at least three replicates.

5. Conclusions

In summary, we identified 29 DREB A4 family members in the poplar genome. They displayed a remarkable conservation in their structural attributes. Transcriptional expression analysis exhibited that most DREB A4 were significantly induced by drought stress. Ectopic expression of PtrDREB4, both in yeast and Arabidopsis, enhanced drought tolerance. Collectively, our findings elucidate the dual roles of poplar DREB A4 genes in regulating plant growth and abiotic stress responses. This knowledge paves a way for further explorations in the realm of plant genetic enhancement and stress tolerance strategies.