PwuWRKY48 Confers Drought Tolerance in Populus wulianensis

Abstract

Populus wulianensis mainly grows in hilly and sloped areas and has strong resistance to adversity. Previous transcriptome studies have shown that a WRKY gene PwuWRKY48 is expression-induced under drought stress. In this study, we aim to characterize the gene’s structure and investigate its role in plant drought resistance. The results show that PwuWRKY48 (1113 bp) belongs to a class IIc WRKY subfamily and it was determined as a nuclear localization protein. The gene promoter region contains a variety of cis-elements in relation to stress resistance. Under drought stress, PwuWRKY48 was expression-induced in leaves and stems, 29.7 and 16.6 times those before treatment, respectively. Overexpressing PwuWRKY48 lines were associated with increased activities of peroxidase (POD) and superoxide dismutase (SOD), 2.5 and 1.6 times higher than those of the wild type. While malondialdehyde content (MDA), superoxide anion radical (O₂^·−), and relative conductivity were decreased by 20%, 30%, and 21.3%, proline and chlorophyll contents increased by 37.5% and 11.2%, respectively. This indicates that PwuWRKY48 efficiently improved the drought tolerance of transgenic plants. PwuWRKY48 can be used as a gene resource for molecular breeding of plant drought resistance.

1. Introduction

WRKY is a kind of transcription factor that functions via regulating the expression level of target genes. It is widely involved in plant growth and development as well as in adversity resistance processes [1,2]. For example, overexpressing Pyrus betulaefolia PbrWRKY53 in Nicotiana tabacum L. increased the expression level of PbrNCED1 and caused vitamin C accumulation [3], while knockdown of PbrWRKY53 down-regulated the expression of PbrNCED1 and reduced drought tolerance. Phyllostachys edulis PheWRKY86 binds to the W-box element in the promoter region of NCED1 to enhance the drought tolerance of plants [4]. PtrWRKY75 from Populus trichocarpa regulates the expression of PAL1 and promotes SA biosynthesis, which increases the accumulation of reactive oxygen species (ROS), reduces stomatal pore size, and enhances drought resistance [5]. CRK5, a cysteine-rich receptor-like protein kinase in Arabidopsis thaliana, is involved in ABA signaling and is jointly negatively regulated by AtWRKY18, AtWRKY40, and AtWRKY60 [6]. PtrWRKY19 could inhibit the transcription of PtoC4H2 and negatively regulate the formation of the secondary wall of the pulp in P. trichocarpa [7].

The expression of transcription factors is also regulated by other proteins and hormones. For example, the PeWRKY41 promoter contains several cis-elements associated with stress resistance [8], resulting in the increased expression of PeWRKY41 under ABA, ethylene, MeJA, salicylic acid, and NaCl stresses. Therefore, it is of great significance to analyze the composition of the transcription factor genes and their promoter elements and to understand the regulation process and gene network that determine their functions.

Populus wulianensis S. B. Liang and X. W. Li is a tree mainly distributed in barren habitats, displaying strong drought resistance [9,10]. Wild populations of P. wulianensis are only distributed on Kunyu Mountain, Zhaohu Mountain, and Jiuxian Mountain in Shandong, China [11]. The number of surviving individuals of this endemic species has been declining because of many threats, which mainly include anthropogenic activities, habitat destruction, and climate change. This species has also been listed in the provincial rare and endangered conservation list in Shandong Province [12]. A previous transcriptome analysis (GEO accession # GSE 252898) showed a WRKY gene significantly enhanced under drought stress in P. wulianensis. Sequence alignment revealed it to be homologous to A. thaliana AtWRKY48, and it was then named PwuWRKY48. In this paper, we aim to investigate the expression model under drought stress and the effects of associated regulatory elements on the promoter and further evaluate its contribution to plant resistance against drought stress through genetic transformation. These results might provide a gene resource for a drought-resistant breeding program and offer more ideas for conservation and utilization of P. wulianensis germplasm.

2. Materials and Methods

2.1. Plant Materials and Drought Treatment

Newly extracted shoots of P. wulianensis with buds in the spring were used as explants and inserted in the bud induction medium (1/2 MS (NH₄NO₃) + 0.05 mg/L naphthalene acetic acid (NAA) + 0.4 mg/L 6-benzylaminopurine (6-BA)) [13]. The differentiated buds were inserted in 1/2 MS rooting medium supplemented with 0.05 mg/L NAA. The culture conditions were 6000–8000 lux light intensity, 16/8 h of light/dark cycle, 25 °C, and relative humidity of 60%–70%.

One-month-old P. wulianensis seedlings were transplanted into pans (7 cm × 7 cm × 7.5 cm) with light substrate (peat:perlite:vermiculite = 3:1:1) and grown in the greenhouse (6000 lux light intensity, 16/8 h of light/dark cycle, 25 °C). Water irrigation was provided once every three days. After one month of growth, the water irrigation was removed for 10 days as a drought treatment. The regular watering was used as a control. The soil was sampled for moisture measurement during the drought stress according to the formula [(fresh soil sample weight − dried soil sample weight)/fresh soil sample weight]. The stems, leaves, and root tissues were also harvested on each day of drought treatment for RNA isolation and physiological index tests.

2.2. Gene Cloning and Vector Construction

The total RNA from P. wulianensis leaves was extracted by a modified hexadecyl trimethyl ammonium bromide (CTAB) method [14]. The 1st-strand cDNA was synthesized using the PrimeScript™ IV 1st Strand cDNA Synthesis Mix Kit (Takara, Dalian, China). Based on the genomic data (https://phytozome-next.jgi.doe.gov/, accessed on 12 September 2020) of P. trichocarpa, PtrWRKY48-specific primers (PtrWRKY48F: 5′-TCACACTCTCACAATGCT-3′: PtrWRKY48R: 5′-ATGTGAACTGGGATCGTCT-3′) were designed. RT-PCR was performed using PrimeSTAR^® Max DNA Polymerase (TaKaRa, Dalian, China). The PCR products were recovered and ligated to the pMD19-T™ vector with a Cloning Kit (Takara, Dalian, China) and transformed into Escherichia coli for sequencing.

For construction of the recombinant expression vector, a pair of primers (PwuWRKY48LCF: 5′-CCCAAGCTTATGAATGAAATGAAGGAGAATAAT-3′: PwuWRKY48LCR: 5′-TGCTCTAGACTACTCCTCTTTAGGCTCCT-3′) were further designed at both ends of the PwuWRKY48 gene with HindⅢ and XbaⅠ cleavage sites, respectively. The full-length gene fragment of PwuWRKY48 was amplified and recovered. Both the gene fragment and the purified expression vector PEZR-(K)-LC plasmid were digested with HindIII and XbaI and ligated with T4-DNA ligase (Takara, Dalian, China) (Figure 1), then transformed into E. coli. The recombinant plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101. The positive clones were identified and used for the subsequent genetic transformation.

2.3. Gene Characterization and Phylogenetic Analysis

To explore the evolutionary relationship of PwuWRKY48, six WRKY members were selected from each subgroup (I, IIa–e, III) of the WRKY family in A. thaliana and P. trichocarpa and phylogenetic analysis was performed. The amino acid sequences of the WRKY genes were obtained from the Phytozome database (http://www.phytozome.com, accessed on 31 October 2023). Multiple sequence alignment was conducted using ClustalW. 2.0.11 A phylogenetic tree was constructed using MEGA 10 with the maximum likelihood method, 1000 repetitions of bootstrap tests, and a JTT matrix-based model. The sequence comparison figure was generated by use of DNAMAN 9.0 software. The theoretical isoelectric point, molecular weight, and hydrophilicity/hydrophobicity of PwuWRKY48 protein were calculated using ProtParam online software (https://web.expasy.org/protparam/, accessed on 31 October 2023).

2.4. Expression Analysis of PwuWRKY48

The total RNA was extracted from each sample of P. wulianensis during drought treatment and reverse transcribed using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa, Dalian, China). cDNA was used as a template for quantitative real-time PCR (qRT-PCR) analysis, and each sample was tested three times. The relative expression of genes was calculated using the 2^−∆∆CT method [15]. The significance of expression difference between the drought-treated sample and control was evaluated using a one-way ANOVA model. Two pairs of fluorescent quantitative primers were designed for PwuWRKY48 (qPwuWRKY48F: 5′-CAAGGAGTTATTATCGTTGC-3′; qPwuWRKY48R: 5′-TGGATGTATGTGTTGACCT-3′) and reference gene Actin7 (qActinF: 5′-CGTACAACTGGTATTGTGT-3′; qActinR: 5′-CTCAGTCAGAATCTTCAT-3′).

2.5. Subcellular Localization of PwuWRKY48

The specific primers were designed for the gene fragment of PwuWRKY48 without termination codon (pNCPwuWRKY48F: 5′-AGTGGTCTCTGTCCAGTCCTATGAATGAAATGAAGGAG-3′; pNCPwuWRKY48R: 5′-GGTCTCAGCAGACCACAAGTCTCCTCTTTAGGCTCC-3′). The product was integrated into the 5′ end of the GFP coding region of the transient expression vector pNC-Amp-GFP-C [16] to construct a 35S::PwuWRKY48::GFP fusion vector by seamless cloning technology.

Tobacco leaf protoplasts were extracted [17]. A PEG-mediated method [18] was used to transfer GFP-fusion vector into the protoplasts. Leaf protoplasts were observed with an Olympus FV1000 (Olympus, Japan) confocal microscope. GFP fluorescence was observed with 473 nm laser excitation and emission between 487 nm and 521 nm, and chloroplast autofluorescence was observed with 559 nm laser excitation and emission between 606 nm and 673 nm.

2.6. Promoter Cloning and Cis-Acting Element Identification

Based on the genome database of P. trichocarpa, specific primer pairs (ProPwuWRKY48F: 5′-TCACACTCTCACAATGCT-3′: ProPwuWRKY48R: 5′-ATGTGAACTGGGATCGTCT-3′) upstream of the PwuWRKY48 gene (approximately 1000 bp from the start codon) were designed. The promoter fragment was amplified with the genomic DNA of P. wulianensis, cloned, and sequenced. The cis-elements in the promoter region were predicted using the online software PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 October 2023).

2.7. Gene Transformation

The positive agrobacterium strains with PwuWRKY48 plasmid were inoculated into YEB medium solution and cultured until OD600 reached 0.5–0.6. After centrifugation, the strains were collected and resuspended in 1/2(NH₄NO₃) MS liquid medium with 100 µmol/L acetosyringone (β-AS). The healthy P. wulianensis leaves were harvested and cut 3–4 times along the main leaf veins, and then soaked in the positive strain solution for 10 min. They were then moved onto solid 1/2(NH₄NO₃) MS medium with 100 μmol/L β-AS in the dark. After 3 days of co-culture, the leaves were transferred onto differentiation medium (1/2(NH₄NO₃) MS + 0.5 mg/L 6-BA+ 0.05 mg/L NAA + 100 mg/L kanamycin + 300 mg/L timentin). The regenerated buds were transferred on rooting medium (1/2 MS + 0.05 mg/LNAA + 100 mg/L kanamycin + 300 mg/L timentin). The induced resistant rooted seedlings were taken from the leaves and cultured again for amplification of leaf-induced adventitious shoots, while the rooted seedlings were transplanted for refining, and after one month of greenhouse culture, the leaves of rooting seedlings were collected for DNA extraction with a M5 Hiper Plant Genomic DNA Kit (Mei5bio, Beijing, China). After PCR testing for PwuWRKY48, the positive plants were used for total RNA isolation, which were then used for expression testing via qRT-PCR.

2.8. Drought Resistance Evaluation of the Transgenic Plants

The overexpressing PwuWRKY48 poplar lines and wild type were vegetatively propagated. The identical clonal plants were transplanted into pans with light substrate (peat:perlite:vermiculite = 3:1:1). After two months of regular management, the poplar plants had water deficiency for 10 d (regular watering was used as a control). The leaves were then harvested for the physiological index tests.

Chlorophyll content of the plants was determined using a chlorophyll meter (SPAD-502-PLUS, Konica Minolta, Japan) [19]. Leaf-staining experiments were carried out with nitrotetrazolium chloride blue (NBT), 3,3′-diaminobenzidine (DAB), and Evans blue, respectively [20]. H₂O₂ content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, proline content, O₂^·− content, and malondialdehyde (MDA) content were measured using Physiological Indicator Determination Kits (Solarbio Science and Technology, Beijing, China). Relative conductivity was determined using a conductivity meter (S230-K, Mettler Toledo, China) [21]. Three replicates for each line/treatment were analyzed. IBM SPSS Statistics 26 software was used for multiple comparisons between the transgenic line and the wild type at significant difference level p = 0.05.

3. Results

3.1. Expression Pattern of PwuWRKY48 Gene in Drought Treatment

Soil moisture was measured during the drought treatment. The separate values were 68.6% (0 d), 65.2% (1 d), 63.2% (2 d), 60.4% (3 d), 57.3% (4 d), 53.8% (5 d), 48.6% (6 d), 40.5% (7 d), 33.2% (8 d), 25.1% (9 d), and 20.8% (10 d). One-month-old P. wulianensis plants grew well in the earlier days of the drought treatment, but the leaves started to wilt at 8 d, and severe wilting occurred at 10 d (Figure 2).

Physiological index tests also showed a great change in the late days of drought treatment compared with that in the earlier days (0–5 days). For example, the relative electrical conductivity of plants changed slightly, 0.56-fold higher at 5 d than that at 0 d [(5 d − 0 d)/0 d]. It increased significantly from 8 d, 2.4-fold higher at 10 d than that at 0 d (Figure 3A). Meanwhile, SOD activity displayed a similar trend, 0.1-fold higher at 5 d than that at 0 d, but 4.1-fold higher at 10 d than that at 0 d (Figure 3B). P. wulianensis sustained drought injury from 7 d.

The PCR tests showed that PwuWRKY48 was expressed in all tissues of stems, roots, and leaves of P. wulianensis (Figure 4A) under normal growth conditions. It had the highest expression value in roots, 1.8 to 4.3-fold higher than those in the stems and leaves, respectively. In the early days of water control (0 d–5 d), the PwuWRKY48 expression model displayed first an upward and then a downward trend in the roots. It reached the peak at 1 d and 2 d of stress treatment, both 2.0-fold higher than those at 0 d (Figure 4B). In the later stress treatment (6 d–10 d), PwuWRKY48 expression displayed a continuous increase. Additionally, PwuWRKY48 had relatively low expression in the stems in the early days of water control, then gradually increasing later. It reached its peak at 10 d with an expression level 17.6-fold higher than that at 0 d (Figure 4C). In leaves, PwuWRKY48 had relatively low expression in the early days of water control, then increasing sharply at 8 d. It reached a peak at 9 d with an expression level 28.7-fold higher than that at 0 d, and then gradually decreased (Figure 4D). In summary, P. wulianensi roots are sensitive to water deficiency, while leaves and stems were somewhat insensitive until real drought stress started.

Comprehensively, the phenotype/physiological performance and PwuWRKY48 expression pattern of P. wulianensis under drought treatment were positively associated, indicating that PwuWRKY48 was involved in plant drought resistance.

3.2. Characterization of PwuWRKY48 Gene and Homologous Analysis

A full-length cDNA fragment of PwuWRKY48 was cloned from P.wulianensis using RT-PCR, with a coding region of 1113 bp (370 amino acids) (GenBank accession # PP101018) (Figure 5). ExPaSy analysis showed that the PwuWRKY48 protein has molecular formula C₁₇₈₅H₂₈₀₃N₅₀₃O₅₈₄S₁₄ with a relative molecular weight of 41.11 kD, theoretical isoelectric point 6.41, and hydrophilic/hydrophobic GRAVY value of −0.743, indicating it to be a hydrophobic protein.

A phylogenetic tree was constructed with the WRKY members in A. thaliana and poplar and PwuWRKY48. As shown in Figure 6A, the PwuWRKY48 protein belongs to the WRKY IIc subfamily and contains a subfamily-specific WRKYGQK heptapeptide sequence and a Cx₄Cx₂₃HxH zinc-finger domain (Figure 6B).

3.3. Identification of the Regulation Elements on PwuWRKY48 Promoter

A promoter fragment of PwuWRKY48 with a length of 1208 bp (GenBank accession # PP101019) was cloned using P. wulianensis DNA as a template (Figure 7). The cis-acting elements in the sequence were determined and showed it contained typical TATA-box and CAAT-box, as well as some adversity-responsive elements such as MYB, ARE, ABRE, CGTCA-motif, and TGACG-motif (Figure 7) and some light-response elements such as GATA-motif, Box 4 and G-Box. Accordingly, PwuWRKY48 expression is in response to light signaling and abiotic stresses.

3.4. Subcellular Localization of PwuWRKY48

The 35S::WRKY48-GFP fusion vector and 35S::GFP empty control, respectively, were transformed into tobacco protoplasts using PEG-mediated transformation. The fluorescence signal showed that the WRKY48-GFP protein was only addressed in the nucleus, but the fluorescent signal of the control covered the whole cell (Figure 8). This indicates that PwuWRKY48 is a nuclear localization protein and might function as a regulator by binding to the promoter region of the target genes.

3.5. Drought Resistance Analysis of P. wulianensis Plants with PwuWRKY48 Overexpression

Transformation of PwuWRKY48 was conducted. Based on DNA tests, seven positive lines (OE1–OE7) were obtained. Gene expression assay showed that OE2, OE3, and OE5 had high expression of PwuWRKY48, 40.2, 70.6, and 91.4 times higher than the wild type, respectively (Figure 9). These three lines were selected for the subsequent resistance evaluation.

The three OE lines were vegetatively propagated. After 2 months of transplanting, three plants from each line with consistent growth were subjected to drought treatment. The results showed that there was no significant morphological difference between the transgenic and wild-type plants under regular growth conditions (Figure 10). At 8 d of drought stress, most leaves of the wild type wilted, while the OE lines grew better with a few basal leaves wilting slightly.

Evans blue staining assays showed that the coloration of the wild-type and OE leaves was similar under regular conditions (Figure 11). However, the coloring area of wild type leaves increased significantly at 8 d of drought stress, while the leaves’ coloring in OE lines increased a little compared to that before drought treatment. Obviously, OE lines had better cell activity and more drought tolerance than the wild type.

Physiological index tests showed that no significant difference was present between the wild-type and OE lines under regular conditions. Along with the drought treatment, all the indexes changed significantly. Among them, the conductivity in the wild type was increased by 28.5% at 8 d of drought stress compared with that at 0 d [(8 d − 0 d)/0 d] and 7.2% in the OE lines (Figure 12A). Meanwhile, MDA content in the wild type increased 1.3-fold and increased 1.0-fold in the OE lines (Figure 12B). Proline content in the wild type increased 1.5-fold and increased 3.2-fold in the OE lines (Figure 12C). Chlorophyll content in the wild type reduced by 17.3% and reduced by 6.2% in the OE lines. Obviously, the OE lines were more tolerant to drought stress than the wild type (Figure 12D).

NBT and DAB staining of plant leaves showed that the wild-type and OE lines performed similarly under regular growth conditions. At 8 d of drought stress, all the lines colored more deeply. Of them, the wild type exhibited significant changes while the OE lines changed a little (Figure 13A). Further measurement of O₂^·− content showed that all were at a low level and no significant difference was found between the wild-type and OE lines under regular growth conditions, but O₂^·− content increased significantly under drought stress in all lines. That in OE lines increased less, 0.7-fold in comparison with the wild type at 8 d of drought stress (Figure 13B). Similarly, SOD and POD activities in both wild-type and OE lines were maintained at low levels under normal conditions and no significant differences were found between them. However, under drought stress, SOD and POD activities increased in all lines. SOD activity was 2.5-fold higher and POD activity was 1.6-fold higher in OE lines than the wild type at 8 d of drought stress (Figure 13C,D). These results reveal that overexpressing PwuWRKY48 could significantly reduce O₂^·− accumulation in leaves under drought stress, which is beneficial for regular metabolic activity in cells.

4. Discussion

4.1. Cis-Regulation of WRKY Expression

WRKY is a large gene family in the plant genome and is involved in plant growth and development and adversity resistance. Each WRKY gene has its specific expression pattern in tissues and organs and under abiotic stress. For example, IbWRKY2 is more highly expressed in the leaves of Ipomoea batatas than that in the stems, hairy roots, fibrous roots, or storage roots [22]. HbWRKY1 is more highly expressed in the latex of Hevea brasiliensis than in the bark, leaves, flowers, or roots [23]. Salt stress could promote the transcription of AcWRKY29, AcWRKY40, AcWRKY48, AcWRKY55, AcWRKY95, and AcWRKY96 in Actinidia chinensis, while it decreased the transcription of AcWRKY38 [24]. RNA-seq results revealed that 61 PtrWRKYs were subjected to biotic and abiotic stresses such as Marssonina brunnea, wounding, cold, and salinity. They were also expressed in different tissues including roots, stems, and leaves [25]. Furthermore, 33 SiWRKYs and 26 SiWRKYs in Sesamum indicum were determined in response to flooding and drought, respectively [26]. In this study, PwuWRKY48 was more likely to be expressed in roots but less in stems. Possibly, the variable expression pattern of WRKY genes is due to its upstream regulators, which bind to the promoter region of the WRKY genes and regulate their expression level. Thus, it is important to understand the regulatory elements affecting WRKY promoters.

Several studies have analyzed the regulatory elements of WRKY gene promoters. For example, PtrWRKYs were widely involved in stress response in P. trichocarpa, in which the promoter region contains various elements associated with stress and defense such as TCA-element, CGTCA-motif, EIRE, ELI-box3, Box-S, WUN motif, ABRE and W-box [25]. The tomato fruit ripening regulator SlRIN was able to bind to the CArG cis-acting element in the promoter region of the SlWRKY35 gene, up-regulate its expression, and enhance carotenoid accumulation [27]. Under non-oxidative conditions, WRKY25 can bind to an element W-box in the WRKY53 promoter and act as a positive regulator of WRKY53 expression, whereas oxidative conditions inhibit the regulatory role of WRKY25 [28]. In this study, we obtained a 1208 bp promoter fragment of PwuWRKY48, containing multiple adversity-related elements MBS, ARE, ABRE, CGTCA-motif, and TGACG-motif, as well as many elements involved in the light-responsive GATA-motif, Box 4, and G-Box. Accordingly, PwuWRKY48 was subjected to various abiotic stress responses such as light signal, drought, and anaerobiosis. These elements coincided with the expression pattern of PwuWRKY48 in stems and leaves under drought stress. It was expected that the associated elements would be identified and the regulatory mechanism for PwuWRKY48 could be understood through yeast one-hybridization and other techniques.

4.2. PwuWRKY48 Gene Advanced Drought Tolerance in Plants via Regulating Target Gene Expression

Several studies have reported that the WRKY genes are widely involved in stress resistance via regulating target gene expression [1]. RNA-seq was assayed for Populus yunnanensis plants with PyWRKY48 overexpression under cadmium stress. Analysis of differentially expressed genes (compared to the wild type) showed that the PyWRKY48 gene could upregulate the expression of the cell wall protein PaGRP, the antioxidant enzyme PaPER, and the heavy metal-related protein PaPHOS. These genes are greatly involved in tolerance to cadmium [29]. WRKY46 is able to bind to a specific W-box region on the VITL1 (vacuolariron transporter like1) promoter and repress VITL1 transcription, which regulates the uptake and translocation of iron in plants under iron deficiency conditions [30]. WRKY7/WRKY11/WRKY17 can inhibit the transcription of bZIP28 by binding to the W-box element in the bZIP28 promoter, and decreased the resistance of A. thaliana to Pseudomonas syringae [31]. Overexpressing AtWRKY40 in A. thaliana can bind to the W-box of the promoter region of ABA signaling regulator genes (ABF4, ABI4, ABI5, and MYB2) and repress their expression [32]. In this study, we obtained PwuWRKY48-overexpressing lines. POD and SOD activity of the plants were significantly increased under drought stress compared with those of the wild type (Figure 12C,D), while O₂^·− and MDA contents were significantly reduced (Figure 12B). This indicates that PwuWRKY48 could reduce the membrane damage caused by ROS. Further detection of target genes is necessary to ascertain the regulatory network of PwuWRKY48 in cells and to understand the mechanism against drought stress.

4.3. WRKY Genes Exhibited Various Resistance to Abiotic Stress

Gene transformation is an efficient way to evaluate the contribution of the genes in stress resistance. CbWRKY27 was overexpressed in Catalpa bungei. Under salt stress, O₂^·− content in the overexpressing line was greatly increased, 1.31-fold compared with the wild-type plant. Meanwhile, H₂O₂ content was reduced to 63.50%, MDA content increased by 17.90%–25.25%, and POD and SOD activity decreased by 52.74% and 18.10%, respectively [33]. MuWRKY3 overexpression in Arachis hypogaea L. showed a 3.5 to 2-fold decrease in MDA content, 1.5- to 2.5-fold increase in proline content, 3–5-fold increase in SOD activity, 3–7-fold increase in APX activity, and 2.3- to 4.4-fold increase in total soluble sugar content compared with wild-type plants under drought stress [34]. In this study, the overexpression of PwuWRKY48 in P. wulianensis under drought stress resulted in increases in POD activity and SOD activity, 2.5- and 1.6-fold higher than the wild type, while MDA and O₂^·− content were 0.2-and 0.3-fold lower, proline and chlorophyll content were 37.5% and 11.2% higher, and relative conductivity was 21.3% lower. This indicates that PwuWRKY48 exhibits strong drought resistance and can be used as a gene resource for molecular breeding programs in plant drought resistance. The various contribution of each WRKY gene to stress resistance is possibly due to their differences in gene sequence and protein structure. More efforts are required in the future to reveal the molecular basis of WRKY genes’ function.

5. Conclusions

The P. wulianensis transcription factor PwuWRKY48 promoter involves multiple adversity-induced inducible elements, which showed a significant up-regulation trend under drought stress and positively regulated the drought tolerance of poplar by increasing the scavenging capacity for ROS, promoting the content of proline, and inhibiting the accumulation of MDA substances. It can be used as a genetic resource for molecular breeding of drought tolerance in plants.