Yazhi Zhao1,2,3†
Zhengquan He1†Lijiao Fan2,3Huan Liu1,2,3Huiling Chen1,2,3Yuying Yang2,3Na Fan1,2,3
Xiaojiao Han2,3
Zhuchou Lu2,3*
Renying Zhuo2,3*- 1Key Laboratory of Three Gorges Regional Plant Genetic & Germplasm Enhancement (CTGU), Biotechnology Research Center, China Three Gorges University, Yichang, China
- 2State Key Laboratory of Tree Genetic and Breeding, Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, China
- 3Zhejiang Key Laboratory of Forest Genetics and Breeding, Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, China
The C2H2 zinc finger protein (C2H2-ZFP) is a large transcription factor (TF) in plants, widely distributed across plants and playing crucial roles in growth, development, and responses to abiotic stress. However, most studies on the C2H2-ZFP gene family have mainly focused on model plants. In this study, we systematically identified the C2H2-ZFP gene family members in Populus euphratica, a tree species with high tolerance to salt and alkali stress, by analyzing gene localizations, conserved motifs, gene structures, and phylogenetic relationships. A total of 67 members of the P. euphratica C2H2-ZFP gene family were identified and were divided into five subfamilies. Promoter analysis revealed numerous cis-acting elements related to development, hormones, and abiotic stress. Both tandem and segmental duplications were identified as the main driving forces behind the expansion of the PeZFP gene family. Expression profiling showed that most PeZFPs exhibit tissue-specific expression patterns and respond to salt stress. Among them, PeZFP38 was strongly induced by salt stress in roots, stems, and leaves, with expression levels increased by 4.3–10.2-fold, 6–10.4-fold, and 28–63.7-fold, respectively. Subcellular localization demonstrated that PeZFP38 is a nuclear protein. Functional assays showed that transient overexpression of PeZFP38 in poplar leaves enhanced salt tolerance, and stable overexpression of PeZFP38 in Arabidopsis thaliana increased biomass (~68% fresh weight), enhanced antioxidant enzyme activities (e.g., SOD activity reached 1.7-fold that of WT), and reduced oxidative damage (~30% MDA decrease). These results suggest that PeZFP38 may play a role in enhancing salt tolerance by integrating ABA signaling with ROS scavenging systems. Collectively, this study systematically deciphers the evolutionary relationships and expression patterns of the C2H2-ZFP family in P. euphratica. For the first time, it functionally identifies the positive regulatory role of PeZFP38 in salt stress response. These findings provide novel genetic resources and a theoretical basis for understanding stress resistance mechanisms and genetic improvement in forest trees.
1 Introduction
Forests represent one of the largest terrestrial carbon reservoir, which is essential for mitigating global warming (Polle and Rennenberg, 2019). Abiotic stress is the primary factor limiting the growth of forest trees, with salt stress being particularly harmful. Salt stress impairs photosynthesis through osmotic stress and ionic toxicity, disrupts metabolic balance, and induces oxidative stress. These effects severely inhibit tree growth (Hao et al., 2021). Understanding the salt tolerance mechanism of forest trees is crucial for selecting stress-resistant species and utilizing them to restore saline soils, which is vital for ecological security (Hao et al., 2021).
To elucidate the mechanisms underlying salt tolerance in forest trees, it is essential to explore the molecular regulatory networks that govern their response to salt stress. Among the numerous molecular components involved, TFs serve as core regulators by activating or repressing downstream target genes to mediate plant abiotic stress responses (Admas et al., 2025; Bokolia et al., 2024). TFs such as NAC (Chen and Xia, 2025), WRKY, bHLH (Filiz and Kurt, 2021), MYB (Wang et al., 2021), and C2H2-ZFP (Han et al., 2020) function as molecular switches within signaling pathways (Liu et al., 2022). Among these, ZFPs constitute the largest family of transcriptional regulators in plants, characterized by finger-like domains containing two cysteine and two histidine residues (Zhou et al., 2022). The ZFP domain is highly conserved and features a common sequence motif, CX2-4CX3FX5LX2HX3-5H, comprising approximately 20–30 amino acids that fold into a spatial structure consisting of two β-sheets and one α-helix (Zhou et al., 2022). ZFPs are classified into types such as C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6, and C8 based on the number and arrangement of cysteine and histidine residues that coordinate zinc ions (Han et al., 2020; Shao et al., 2023). In plants, the zinc finger domain of C2H2-ZFPs typically contains a highly conserved QALGGH motif, which may play a role in regulating plant-specific biological processes (Pu et al., 2021).
The C2H2-ZFP gene family is widespread across the plant kingdom and involved in a wide range of biological functions. These encompass key aspects of plant growth, development, and adaptive reactions to environmental challenges like drought, high salinity, and cold (Nikraftar et al., 2024). In model plants like Arabidopsis thaliana (176) (Lu et al., 2024), tomato (Solanum lycopersicum, 99) (Zhao et al., 2020b), and poplar (Populus trichocarpa, 109) (Liu et al., 2015), the family size, evolutionary relationships, and functions of several members have been well characterized. For example, the first plant C2H2 gene, TAZ, identified in petunias, is involved in petal development, while ZFP5 in A. thaliana regulates ethylene signaling and root hair development (Zhang et al., 2022a). In Arabidopsis, AtSIZ1 enhances salt tolerance through the regulation of ionic homeostasis and osmotic balance (Han et al., 2019). Studies in apple (Malus domestica) have revealed that ZAT5 acts as a negative regulator of salt tolerance by enhancing plant sensitivity to salinity stress (Wang et al., 2022a). In P. euphratica, PeSTZ1 enhances cold tolerance by regulating antioxidant enzyme gene expression (He et al., 2019). In rice, overexpression of OsZFP15 promotes ROS production and compromises cellular oxidative stress tolerance, yet simultaneously improves plant performance under salinity and drought conditions (Wang et al., 2022b). Conversely, in banana, overexpression of MaC2H2-2 and MaC2H2-3 leads to a suppression of the signaling pathway associated with cold stress (Han and Fu, 2019).
As a keystone species of desert riparian forests, P. euphratica possesses exceptional salt and drought tolerance, serving as an ideal system for studying stress resistance mechanisms in trees (Jia et al., 2020). P. euphratica can survive under extreme environmental conditions through mechanisms such as ion homeostasis, reactive oxygen species (ROS) scavenging, and cell wall modification (Niu et al., 2024). Comparative genomic studies have revealed significant expansion of stress-related gene families in its genome (Jia et al., 2020), enhancing ion regulation (e.g., sodium exclusion and potassium accumulation) and maintaining water balance under stress conditions (Zhao et al., 2020a). These results suggest adaptive evolution of its transcriptional regulatory networks.
Current studies on C2H2-ZFPs have primarily focused on model species such as A. thaliana and rice. However, systematic identification, expression profiling, and functional validation of this family remain lacking in P. euphratica. In this study, we systematically identified the evolutionary characteristics and expression patterns of the C2H2-ZFP gene family in P. euphratica based on the whole-genome sequence. Furthermore, we focused on a candidate gene, PeZFP38, whose promoter is enriched with stress-responsive cis-acting elements. PeZFP38 belongs to one subfamily, which contains P. euphratica-specific clades, and is rapidly and persistently induced in roots, stems, and leaves under salt stress. Heterologous overexpression assays confirmed that PeZFP38 significantly enhances salt tolerance in both 84K poplar and Arabidopsis. These findings provide a framework for elucidating the molecular basis of adaptation to environmental stress in P. euphratica and offer valuable genetic resources for forest tree improvement (Ndayambaza et al., 2025).
2 Materials and methods
2.1 Plant materials and growth conditions
The plant materials used in this experiment were all obtained from and preserved in the Laboratory of Forest Genetics and Breeding, Subtropical Forestry Research Institute, Chinese Academy of Forestry (Hangzhou, Zhejiang). P. euphratica (desert poplar) was selected as a natural seedling clonal line. Young shoots of P. euphratica were collected from a forest in Jiuquan, Gansu Province. Sterilized stem segments were cultured on rooting medium to generate aseptic seedlings, which were used for subsequent experiments. 84K poplar (Populus alba × Populus glandulosa) was a hybrid first-generation clonal line provided by the Chinese Academy of Forestry. Both P. euphratica and 84K poplar were cultured aseptically in vitro under constant conditions of 24°C, 16 hours (h) light/8 h dark photoperiod, and 50%–60% relative humidity. The wild-type (WT) A. thaliana used was the Columbia ecotype (Col-0). A. thaliana seeds were stored at -4°C and subsequently germinated on half-strength Murashige and Skoog (MS) solid medium (M519, PhytoTech Labs, US) before cultivation. The materials used in this experiment comprised 3-month-old aseptic P. euphratica seedlings, 1-month-old aseptic 84K poplar seedlings, and 2-week-old/1-month-old A. thaliana seedlings. All materials were in consistent growth condition and free from disease.
2.2 Methods
2.2.1 Identification of C2H2-ZFP gene family in P. euphratica
P. euphratica genome and protein sequences were downloaded from the China National Center for Bioinformation (https://ngdc.cncb.ac.cn/omix/). Genomic data for A. thaliana and P. trichocarpa were obtained from the Ensembl Plants database (https://plants.ensembl.org/index.html). To comprehensively identify all C2H2-ZFP gene family members in P. euphratica, a dual strategy was employed. First, the hidden Markov model (HMM) profile corresponding to the C2H2-ZFP domain (PF00096) was acquired from the Pfam database (http://www.ebi.ac.uk/interpro/). This profile was used as a query to search for the C2H2-ZFP genes in the P. euphratica genome using TBtools-II software (version 2.371) (Chen et al., 2020). Second, 173 known C2H2-ZFP proteins from A. thaliana were used as query sequences for BLASTP searches against the P. euphratica protein database with an E-value < e-5. The results from both searches were merged, and redundant sequences were removed. All candidate proteins were then validated for the presence of C2H2-ZFP domains using SMART and the NCBI Batch CD-Search tool.
2.2.2 Chromosomal location and characterization analysis
The 67 identified PeZFP genes were renamed according to their physical positions on the chromosomes of P. euphratica. The Protein Parameter Calculator function in TBtools-II software was used to determine the number of amino acids, molecular weight, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity for each PeZFP protein. The subcellular localization of the identified proteins was predicted using the WoLF PSORT (https://wolfpsort.hgc.jp) online tool.
2.2.3 Phylogenetic analysis
An unrooted phylogenetic tree was generated using 67 full-length PeZFP proteins from P. euphratica and 173 full-length AtZFP proteins from A. thaliana. The maximum likelihood (ML) method, implemented in MEGA-X, was used with 1000 bootstrap replicates. The resulting phylogenetic tree was further classified and visualized using the iTOL online platform (https://itol.embl.de/).
2.2.4 Conserved motif and gene structure analysis
The conserved motifs of the PeZFP proteins were predicted utilizing the MEME program (version 5.5.8) (https://meme-suite.org/meme/), with the maximum number of motifs constrained to ten. Exon-intron structures of PeZFP genes were derived from the genome annotation file of P. euphratica. The phylogenetic tree, conserved motifs, and gene structures of PeZFPs were subsequently integrated and visualized using TBtools-II software (Chen et al., 2020).
2.2.5 Promoter cis-acting elements analysis
The 2-kb upstream promoter sequence of each PeZFP gene, relative to the translation start site, was extracted using TBtools-II software. The sequences were analyzed using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify cis-acting regulatory elements (CREs). The types and frequencies of CREs were summarized and visualized using Microsoft Excel and the Gene Structure Viewer tool in TBtools-II.
2.2.6 Collinearity and gene duplication event analysis
The MCScanX module in TBtools-II was used to analyze collinearity relationships within the PeZFP gene family. The results were visualized using the Advanced Circos tool in TBtools-II. Interspecific collinearity between P. euphratica and A. thaliana or P. trichocarpa was visualized using the Dual Synteny Plot tool in TBtools-II, respectively. The ratio of non-synonymous (Ka) to synonymous (Ks) nucleotide substitutions for duplicated gene pairs was calculated using the Simple Ka/Ks Calculator to determine selection patterns.
2.2.7 Expression pattern analysis under salt stress
P. euphratica seedlings were hydroponically cultured in 1/2 Hoagland nutrient solution (NSP1020, Coolaber, China) for 3 days (d) before salt treatment. Salt treatment was performed by adding 300 mM NaCl (Ge et al., 2022) to the nutrient solution. All samples of roots, stems, and leaves were harvested at 0 h, 6 h, 12 h, 1 d, 4 d, and 7 d (Li et al., 2024) after salt treatment for transcriptome sequencing. RNA-seq libraries were constructed and sequenced on an Illumina platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). Clean reads were aligned to the P. euphratica v2.0 genome and quantified following standard pipelines (Li and Dewey, 2011; Wang et al., 2009). Raw data are available in the China National Center for Bioinformation database under accession PRJCA050032. Expression levels were measured in TPM value. Expression levels of PeZFPs across time points and tissues were organized and quantified in Microsoft Excel. Data with no detectable expression were excluded from statistical analysis. Three biological replicates were used. Data were normalized using Z-score standardization by column or by row before clustering. Heatmaps were generated using TBtools for visualization.
2.2.8 Subcellular localization
The CDS sequence of the PeZFP38 (with the stop codon removed) was cloned into the pBI121-GFP vector via XbaI and KpnI sites to construct the fusion expression vector pBI121-PeZFP38-GFP. The primers used are listed in Supplementary Table S5. Using P. euphratica cDNA as the template, the product was amplified with Phanta Max Master Mix (P525-03, Vazyme, China). After gel purification, ligation, and transformation, positive clones were verified by sequencing (Shangya Bio, Hangzhou, China). The recombinant construct was transformed into the Agrobacterium strain GV3101 (CC96304, TOLOBIO Biotech, China). Positive Agrobacterium cultures were grown in LB medium containing the appropriate antibiotics until OD600 reached 0.8. Cells were collected by centrifugation and resuspended in infiltration buffer (containing 10 mM MES, 20 mM MgCl2, and 200 μM acetosyringone) to an OD600 of 1.0, and incubated at room temperature for 2 h. For infiltration, bacterial suspensions containing the target or control vector were mixed at a 1:1 ratio with the nuclear marker p2300-35S-D53-mCherry. The mixed suspension was then infiltrated into the abaxial side of Nicotiana benthamiana leaves. After infiltration, the plants were kept in the dark for 12 h to reduce fluorescent protein photodegradation, then transferred to normal light conditions for 36 h to promote protein folding and accumulation. The subcellular localization of the fusion protein was examined using a confocal laser scanning microscope (LSM900, Zeiss, Germany).
2.2.9 Transient PeZFP38 gene expression analysis in poplar leaves
Healthy one-month-old sterile seedlings of 84K poplar with uniform growth were used in the transient expression experiment. Leaf discs (6 mm in diameter) collected from the 2nd to 4th leaves at the morphological apex. These discs were then transiently transformed via Agrobacterium GV3101 carrying either the experimental plasmid pBI121-PeZFP38-GFP or the control plasmid pBI121-GFP, as referenced in (Chen et al., 2025; Lin et al., 2017). The transformed leaves were first incubated on 1/4 MS solid medium under dark conditions for 2 d, and then transferred to 1/4 MS liquid medium containing 0, 100, 300, or 500 mM NaCl for 7 d. Based on previous studies (Chen et al., 2025; Lei et al., 2024; Li et al., 2024), the NaCl concentration gradient was designed to cover a range from mild to extreme salt stress. Chlorophyll content was measured using a microplate reader (SpectraMax iD5, MD, USA) as previously described (Tyagi et al., 2023) to evaluate the effect of salt treatment. All experiments included at least three biological replicates.
2.2.10 Overexpression of PeZFP38 in A. thaliana
The recombinant plasmid pBI121-PeZFP38-GFP was introduced into A. thaliana using the floral dip method (Ali et al., 2024) with Agrobacterium strain GV3101. Three independent T1 transgenic lines confirmed by genomic PCR and identified as high-expression lines by Quantitative Real-Time PCR (qRT-PCR) were selected and propagated to obtain homozygous T3 plants for all subsequent experiments. For the salt stress plate experiments, 7d seedlings grown on 1/2 MS medium were transferred to medium supplemented with 0, 50, 100, or 150 mM NaCl (Ma et al., 2023; Qu et al., 2023). After 7 d of growth, biomass and root length were measured. For the hydroponic salt stress assay, 20d seedlings cultured in 1/2 Hoagland nutrient solution were treated with 100 mM NaCl for 7 d (Qu et al., 2023), after which growth parameters and antioxidant enzyme activities were analyzed (Deng et al., 2024). All experiments included at least three biological replicates. Histochemical staining with nitroblue tetrazolium chloride (NBT) and 3,3′−diaminobenzidine (DAB) was performed to detect the accumulation of ROS in leaves (He et al., 2020).
2.3 Statistical analysis
All data were statistically analyzed using SPSS (version 27.0) software and visualized using GraphPad Prism (version 9.5.0) software. Data represent the mean ± standard deviation (SD) from at least three independent biological replicates. Before conducting formal statistical tests, the normality of data distribution and homogeneity of variances were verified to satisfy the assumptions for parametric tests. Difference between two groups was assessed by two-tailed unpaired Student’s t-test with p < 0.05. The significance of differences between multiple groups was evaluated by one-way ANOVA followed by LSD test (p < 0.05).
3 Results
3.1 Identification of C2H2-ZFP gene family members in P. euphratica
Using BLASTP and HMM searches for the C2H2-ZFP domain in the P. euphratica genome database, a total of 67 C2H2-ZFP genes were identified. These genes were sequentially renamed PeZFP1 to PeZFP67 based on their chromosomal locations. These genes were distributed across chromosomes 1 to 19 of P. euphratica, with no genes located on chromosomes 15 and 16. Chromosomes 1 and 3 contained the highest numbers of PeZFPs, while only one gene was detected on chromosomes 7, 8, and 9 (Figure 1).
Figure 1. Chromosomal distribution of PeZFPs in P. euphratica. The lengths of the 17 chromosomes are represented on the left in megabases (Mb). Black lines indicate the precise locus of each gene on its corresponding chromosome, while the color gradient along the chromosomes represents gene density.
Analysis of the physicochemical characteristics of the PeZFP proteins is summarized in Supplementary Table S1.The amino acids lengths ranged from 126 to 593, averaging 310. Corresponding molecular weights ranged from 14.67 kDa to 67.96 kDa, with an average of 34.17 kDa. The theoretical isoelectric point showed a spectrum from 4.87 to 9.76, averaging 7.73. The instability index varied between 39.27 and 74.29, averaging 57.08. Unlike PeZFP20, most PeZFPs were predicted to be unstable. The aliphatic index ranged from 40.59 to 86.49, with an average of 60.57. The grand average of hydropathicity varied from −1.202 to −0.134, averaging of −0.72. Subcellular localization predictions indicated that all PeZFPs may localize to the nucleus, which is consistent with the typical characteristics of C2H2-ZFP TFs (Huang et al., 2024).
3.2 Phylogenetic analysis of PeZFPs in P. euphratica
To elucidate the evolutionary relationships and functional diversification of PeZFPs, an unrooted phylogenetic tree was constructed using the maximum likelihood (ML) method based on 67 PeZFP protein sequences and 173 AtZFP protein sequences. AtZFP proteins were named based on their chromosomal locations in A. thaliana. Based on their branch clustering patterns, protein sequence similarities, and previously established classifications (Arrey-Salas et al., 2021; Zhou et al., 2024), the PeZFPs were classified into five subfamilies (I, II, III, IV, V), containing 11, 15, 10, 18, and 13 members, respectively (Figure 2). Key branching nodes received strong bootstrap support (≥70%, represented by color gradients), confirming the reliability of the classification.
Figure 2. Phylogenetic tree of the PeZFP gene family in P. euphratica and A. thaliana. The unrooted tree was constructed using the ML method based on PeZFP and AtZFP protein sequences. The tree is divided into five subfamilies, each highlighted with a distinct color. Branches are annotated with ML bootstrap values (≥70%). AtZFPs are denoted by solid purple circles, whereas PeZFPs are indicated by solid green circles.
Phylogenetic analysis revealed that PeZFPs and AtZFPs frequently formed cross−species mixed branches within each subfamily. This pattern was particularly evident in subfamily I, where the two groups were closely nested, indicating that these subfamilies likely originated prior to species divergence and may have retained conserved functions. Notably, subfamilies II–V contained several clades consisting exclusively of P. euphratica members, suggesting lineage−specific expansion or functional specialization during adaptation to arid and saline environments.
3.3 Conserved motif and gene structure analysis
Phylogenetic analysis based on all PeZFP protein sequences revealed that these proteins can be classified into five subfamilies (I–V) according to evolutionary relationships and sequence similarity (Figure 3A). These subfamilies correspond to subfamilies IV, V, II, III, and I in the phylogenetic tree shown in Figure 2. To further elucidate the potential functions of PeZFPs, conserved motifs and gene structures were analyzed. Ten conserved motifs were identified among 67 PeZFP proteins (Figure 3B).
Figure 3. Conserved motifs and gene structure of PeZFPs in P. euphratica. (A) Phylogenetic tree of PeZFPs, with distinct subfamilies represented by differently colored blocks. (B) Conserved motif compositions of PeZFPs, ten identified motifs are illustrated by distinct color-coded boxes. (C) Gene structure organization of PeZFPs, where exons are shown in yellow, untranslated regions in green, and introns as black straight lines. The scale bar represents gene length in kilobases (Kb).
The number and arrangement of motifs were generally consistent within each subfamily, indicating functional conservation among members of the same subfamily. Except for PeZFP24, PeZFP53, and PeZFP63, all members contained motif 1, indicating that motif 1 was a highly conserved and potentially essential core element of the PeZFP gene family in P. euphratica. Notably, motif 1 and 2 contained the C2H2-ZFP sequences and the plant-specific motif QALGGH. Motif analysis revealed clear classification patterns, based on characteristic motifs, family members could be categorized into Q-type and M-type (Supplementary Table S2). Specifically, a total of 54 members (80.6%) contained the QALGGH motif and were classified as Q-type C2H2-ZFPs. The distribution of the Q-type showed subfamily specificity. All members of subfamilies I and II (excluding PeZFP25) contained one such motif, most members of subfamilies III and IV contained one or two such motif. The majority of members in subfamilies I–IV (e.g., those containing motif 6) possessed the characteristic EAR motif (LxLxL or DLNxxP), which typically conferred transcriptional repression activity (Wang et al., 2020). In contrast, members of subfamily V, which completely lacked this motif, were classified as M-type C2H2-ZFPs and generally did not contain an EAR motif. These results indicate that despite clear evolutionary divergence among the five subfamilies, protein sequences and potential function remain relatively conserved within each subfamily.
Gene structure analysis showed that 59.7% of the genes lack introns, while the rest contain 1 to 5 introns of varying lengths (Figure 3C). Furthermore, the absence of UTRs in 28 genes may stem from the incomplete nature of genome annotation, a limitation commonly encountered in genome-based studies that typically does not compromise coding sequence analysis. Exon–intron patterns were similar within subfamilies. For example, most members of subfamily II contained no introns and exhibited the simplest structure. In contrast, members of subfamily V, which lack the QALGGH motif, mostly contained multiple introns and displayed more complex structures. Such structural differences likely reflect distinct evolutionary histories involving gene duplication and intron gain/loss events, leading to unique structural features and potential regulatory complexity in each subfamily.
3.4 Promoter cis-acting elements analysis
To further elucidate the potential regulatory mechanisms of PeZFPs in plant biological processes, the 2000 bp upstream promoter regions of all PeZFPs were analyzed. The results were arranged according to the phylogenetic tree (Figure 4A). A total of 18 types of CREs were identified, which were categorized into three major groups, such as stress response, plant development, and phytohormonal response. The distribution of CREs in the promoter regions exhibited a non-random clustering pattern, where specific types of elements tended to aggregate together, potentially forming synergistic transcriptional regulatory modules (Figure 4B).
Figure 4. Distribution and number of CREs in PeZFP promoters of P. euphratica. (A) Phylogenetic tree of PeZFPs, with distinct subfamilies represented by differently colored blocks. (B) Distribution of CREs in PeZFP promoters. Different colored boxes represent different types of elements. (C) Number of CREs in PeZFP promoters. Darker red indicates a higher abundance of elements. (D) The histogram depicts the abundance of CREs across three major functional categories. The bars correspond to stress response (red), development (blue), and hormone response (yellow).
Analysis revealed significant differences in the composition and abundance of CREs among different evolutionary subfamilies (Figures. 4C, D). This pattern was consistent with their phylogenetic relationships, characteristic motifs, and gene structural complexity. Specifically, subfamily II showed a relatively high proportion of development-related elements (e.g., metabolism expression and circadian control element), implying that its members may be primarily involved in the basal regulation of plant growth and development. In contrast, subfamilies III and IV were significantly enriched in stress- and hormone-responsive elements, with the anaerobic induction element ARE and the ABA-responsive element ABRE being the most prominent. This suggests that these two subfamilies may play core roles in mediating abiotic stress signal transduction (e.g., drought and hypoxia) and hormone-dependent stress responses. Subfamilies II (with simple gene structures, mostly Q-type) and V (with complex gene structures, mostly M-type) had more diverse and balanced CRE compositions, encompassing all three categories of elements. This indicates that their members may possess broader regulatory plasticity in biological functions.
Except for PeZFP36 and PeZFP54, all PeZFP promoters contained stress-related elements, including those involved in responses to cold, drought, wounding, and hypoxia. Results suggest that these CREs may play potential roles in the adaptation of P. euphratica to abiotic stresses. Among all CREs, ARE, ABRE, CGTCA-motif, and MBS were the most abundant types, which are associated with hypoxia, ABA, methyl jasmonate, and drought responses, respectively (Biłas et al., 2016). These findings highlight these signaling pathways as the core components of the PeZFPs regulatory network in P. euphratica. For instance, PeZFP19, which contains multiple ARE, LTR, and MBS elements, and PeZFP24, which has the largest number of total CREs, are proposed as key candidate regulators of stress responses. Additionally, several elements were found to cooperate in the same regulatory pathways. For example, AuxRR-core, TGA-box, and TGA-element are associated with auxin responses, while P-box, TATC-box, and GARE-motif are all implicated in the regulation of gibberellin responses. This implies that PeZFPs may enhance the sensitivity and amplitude of plant responses to environmental signals through element synergy, thereby fine-tuning hormone demands and stress responses across different growth and developmental stages. Collectively, these complex variations in promoter architectures not only reveal the regulatory basis underlying the functional diversification of PeZFPs but also provide critical clues for interpreting their differential expression patterns in various tissues or under distinct stress conditions.
3.5 Collinearity and gene duplication event analysis
To elucidate the expansion mechanism and evolutionary drivers of the PeZFP gene family, we performed an integrated analysis of duplication events, selection pressure, and interspecific synteny. Tandem duplication and segmental duplication are two primary mechanisms driving gene family expansion (Huang et al., 2022). A total of 38 segmental duplication events and 8 pairs of tandem duplication events were identified (Figure 5), indicating that both types of duplication contributed to its expansion. Notably, tandemly duplicated genes (e.g., PeZFP12/13, PeZFP32/33) often clustered within the same phylogenetic clade or adjacent genomic regions, potentially facilitating the rapid formation of local gene clusters and functional specialization within subfamilies.
Figure 5. Intraspecific collinearity analysis of PeZFP genes in P. euphratica. The gray lines in the background denote collinearity pairs within the species, whereas the red lines signify the PeZFP gene pairs derived from segmental duplication events. The outermost circle displays the chromosome number, and the line map and heat map illustrate gene distribution and density along the chromosomes.
Analysis of Ka/Ks ratios for duplicated gene pairs (Supplementary Table S3) revealed that most values were significantly less than 1 (ranging from 0.12 to 0.62). Results indicated that this family has been under strong purifying selection (Alam et al., 2022) during evolution to maintain the zinc-finger structure and functional constraints. However, some gene pairs (e.g., PeZFP14/23) showed relatively higher Ka/Ks values, suggesting they may have undergone functional divergence. This observation aligns with their distribution in distinct phylogenetic subclades and differences in CRE composition.
Interspecific synteny analysis further revealed the evolutionary conservation and specificity of PeZFPs. A total of 173 collinear gene pairs were identified between P. euphratica and P. trichocarpa, far more than the 81 pairs identified between P. euphratica and A. thaliana (Figure 6). These results suggest that a closer phylogenetic relationship between the two Populus species. Most conserved syntenic genes (e.g., PeZFP16, PeZFP27), which maintained orthologous relationships with both P. trichocarpa and Arabidopsis, contained typical QALGGH and EAR motifs, possessed simple gene structures, and were widely distributed across subfamilies I–IV. Their sequences and functions are likely stable and may be involved in fundamental transcriptional regulation. In contrast, Populus-specific retained gene pairs (e.g., PeZFP40, PeZFP41 and their homologs) were found only within Populus and absent in synteny with Arabidopsis. These genes may have undergone functional differentiation, providing a potential genetic basis for the adaptation of Populus species, such as P. euphratica, to specific habitats. Together, these results demonstrate that the evolution and functional diversification of the PeZFP gene family were shaped by the combined effects of duplication events, purifying selection, and species-specific adaptation.
Figure 6. Collinearity analysis of ZFPs in P. euphratica, P. trichocarpa and A. thaliana. Purple, cyan, and brown represent the chromosomes of P. trichocarpa, P. euphratica, and A. thaliana, respectively. Gray lines in the background indicate all collinear gene pairs among the three species, whereas blue lines connecting distinct chromosomes highlight collinear ZFP gene pairs between P. euphratica and the other two species.
3.6 The expression patterns of PeZFPs in various tissues and under salt stress
Based on the analysis of promoter CREs, PeZFPs contained numerous stress-response elements. To further investigate their expression patterns and potential association with CREs, the expression of 67 PeZFPs in roots, stems, and leaves using transcriptome data of P. euphratica under salt treatment were analyzed. Heatmap were generated using TBtools to visualize the expression patterns (Figure 7).
Figure 7. Expression profiles of PeZFPs in various tissues of P. euphratica under salt stress. (A) Tissue-specific expression patterns of PeZFPs under normal condition. (B–D) Dynamic expression patterns of PeZFPs in roots (R), stems (S), and leaves (L) under 300 mM NaCl stress.
Expression profiling revealed significant tissue-specific expression and differential responses to salt stress among PeZFPs. Under normal conditions, most genes were expressed in roots, stems, and leaves of P. euphratica, indicating their functional diversity (Figure 7A). Some PeZFPs showed no detectable expression. Specifically, four members (PeZFP3, PeZFP36, PeZFP57, PeZFP66), three members (PeZFP3, PeZFP4, PeZFP57), and two members (PeZFP3, PeZFP57) were highly expressed in roots, stems, and leaves, respectively (Supplementary Table S4). Generally, PeZFP3 and PeZFP57 were constitutively highly expressed across all three tissues (Figure 7A). Both genes contain the conserved QALGGH motif, and their promoter regions are enriched with various hormone-responsive and basal regulatory elements, suggesting their potential roles in fundamental physiological processes. Under salt stress, about half of the genes exhibited a trend of downregulation across tissues (Figures 7B–D). Five genes (PeZFP4, PeZFP9, PeZFP33, PeZFP55 and PeZFP57) were markedly down-regulated, whereas four genes (PeZFP15, PeZFP22, PeZFP38 and PeZFP50) were up-regulated.
Further analysis linking expression patterns with phylogenetic subfamilies and CRE compositions revealed significant correlations. Genes in subfamily IV generally showed low expression under normal conditions. However, under salt stress, certain genes, such as PeZFP38, exhibited significant and sustained upregulation across all three tissues. The expression induction ranged from 4.3- to 10.2-fold in roots, 6- to 10.4-fold in stems, and 28- to 63.7-fold in leaves. This gene aligns with the strong enrichment of stress-responsive elements such as ARE and MBS in the promoters of this subfamily, indicating that subfamily IV may play a central role in salt stress signaling. In contrast, some highly expressed Q-type genes under normal conditions, such as PeZFP57, was significantly downregulated after salt treatment. Their promoters contain both developmental-related elements and a limited number of stress-responsive elements, implying that their functions may be more biased toward growth regulation.
Salt stress response analysis showed that different genes exhibited distinct temporal and spatial expression patterns, including rapid, sustained, or delayed responses. For example, PeZFP22 and PeZFP38 were rapidly induced and maintained high expression across roots, stems, and leaves, representing core stress-responsive genes. In contrast, genes such as PeZFP61 were induced only in specific tissues or at certain time points, reflecting regulatory complexity. These differential expression trends are likely determined by the type and combination of CREs in their promoters. For instance, rapidly induced genes often harbor dense clusters of stress-related elements such as ARE and ABRE. Collectively, PeZFPs exhibit distinct transcriptional regulatory patterns across tissues. Most PeZFP members were induced by salt stress, implying their potential involvement in the salt stress responses.
3.7 Subcellular localization of PeZFP38
To investigate the response of PeZFPs to abiotic stress, particularly salt stress, we selected the gene PeZFP38, which is significantly induced by salt stress based on transcriptome results, for study. Previously, subcellular localization of PeZFPs was predicted using TBtools, which indicated that all PeZFPs had the highest scores for nuclear localization. Specifically, PeZFP38 scored 14 for nuclear localization, with no notable scores for other compartments. Based on this prediction, the recombinant vector pBI121-PeZFP38-GFP was constructed, with vector pBI121-GFP serving as a control.
Both constructs were co-transformed into Agrobacterium GV3101 together with the nuclear marker vector p2300-35S-D53-mCherry. After adjusting the bacterial suspension to an OD600 of 1.0, we performed transient co-expression in N. benthamiana leaves by infiltration. The leaves were incubated in darkness for 12 h, followed by 36 h under normal light conditions, after which fluorescence signals were observed using a laser confocal microscope. The results showed that the PeZFP38-GFP and mCherry signals completely overlapped within the nucleus, whereas the GFP signal in the control was detected throughout the entire cell (Figure 8). Therefore, PeZFP38 is a nuclear-localized protein, providing subcellular evidence for its function as a TF involved in the salt stress response, supporting a molecular mechanism whereby it mediates plant salt adaptation through transcriptional regulation within the nucleus.
Figure 8. Subcellular localization of PeZFP38 in N. benthamiana epidermal cells. pBI121-GFP indicates the control. The confocal images of mCherry fluorescence (red), GFP fluorescence (green), bright-field, and merged-field. Bar = 20 μm.
3.8 Transient overexpression of PeZFP38 enhanced the salt tolerance of poplar leaves
To investigate the function of PeZFP38, we conducted transient overexpression assays in leaves of the fast-growing poplar variety 84K, as the transient transformation system for P. euphratica itself has not yet been established. This approach preliminarily assessed the role of PeZFP38 in salt tolerance. Successful introduction of PeZFP38 was confirmed by genomic DNA analysis, and its expression level was about 8-fold higher than in the empty-vector control (Supplementary Figure S1), verifying an effective transient transformation system. Under salt treatment, leaves exhibited progressively severe chlorosis and wilting with increasing NaCl concentration (Figure 9A). Compared with leaves transformed with the empty vector, those overexpressing PeZFP38 showed significantly reduced chlorosis at the same salt concentrations, indicating that PeZFP38 may enhance the tolerance of plant to salt stress.
Figure 9. Transient overexpression of PeZFP38 enhances salt tolerance in poplar leaves. (A) Phenotypes of poplar leaves transiently transformed with PeZFP38-OE and empty vector after 7 d of NaCl treatment. (B) Total chlorophyll content of leaves with PeZFP38-OE and empty vector after 7 d of NaCl treatment. Statistical significance was assessed employing the statistical T-test (**p < 0.01). Bar = 1 cm.
To further quantify these phenotypic differences, total chlorophyll content was measured (Figure 9B). Under 100 mM NaCl treatment, chlorophyll content in PeZFP38-overexpression (OE) leaves was approximately 1.2-fold higher than those in control. Under 300 mM NaCl treatment, it remained significantly higher (about 1.3-fold of the control). Even under 500 mM NaCl stress, chlorophyll in the PeZFP38-OE group was still 1.8-fold higher than in controls. Although chlorophyll declined with rising salt concentration in all treatments, overexpression of PeZFP38 consistently alleviated chlorophyll degradation. These results indicate that PeZFP38 helps maintain chlorophyll stability and positively regulates salt tolerance in poplar.
3.9 Overexpression of PeZFP38 improved salt tolerance in A. thaliana
To further verify the role of PeZFP38 in plant stress resistance, the overexpression vector pBI121-PeZFP38-GFP was introduced into A. thaliana via Agrobacterium-mediated transformation. Positive transgenic lines were confirmed by PCR amplification of the target gene (Supplementary Figure S2) and by assessing expression levels using qRT-PCR (Supplementary Figure S3). Three lines (OE2, OE5, and OE6) showing higher relative expression levels were selected for subsequent functional analyses.
Plate assays demonstrated that PeZFP38 overexpression significantly improved salt tolerance. On medium containing 50, 100, or 150 mM NaCl, WT plants exhibited severe chlorosis and wilting, whereas the growth inhibition of PeZFP38-OE lines was markedly less pronounced (Figure 10A). Under 50 mM NaCl treatment, the root length increment of PeZFP38-OE lines was significantly greater than that of WT, with an average increase of approximately 24.6%. Under 100 mM NaCl treatment, the fresh weight of PeZFP38-OE lines showed a significant average increase of about 68.2% compared to WT (Figures 10B, C). Although the growth advantage of some PeZFP38-OE lines did not reach statistical significance across all salt concentrations, they consistently performed better than WT.
Figure 10. Overexpression of PeZFP38 in A. thaliana increases root length increment and fresh weight under salt stress in plate culture. (A) Phenotypes of PeZFP38-OE plants and WT under normal conditions and after salt treatment. Bar = 1 cm. (B) Root length increment. (C) Fresh weight. Data represent the mean ± standard deviation (SD) from at least three independent biological replicates. Statistical significance was determined using one-way ANOVA (*p < 0.05; **p < 0.01). The same convention applies to the following figures.
Hydroponic experiments further confirmed these findings. Under salt stress, the growth of PeZFP38-OE plants was significantly better than that of WT plants (Figure 11A), whereas WT plants growth was severely inhibited, showing yellowing and partial abscission. PeZFP38-OE lines showed a significantly smaller reduction in root length compared to WT, and their fresh weight accumulation averaged approximately 2.1 times that of WT (Figures 11B, C). Physiological analysis indicated enhanced antioxidant capacity in PeZFP38-OE plants. Under salt stress, the activities of superoxide dismutase (SOD) and catalase (CAT) in PeZFP38-OE leaves reached approximately 1.7-fold and 1.4-fold, respectively, of the levels in WT (Figures 11D, E). Peroxidase (POD) activity also showed an increasing trend (Figure 11F). Correspondingly, the malondialdehyde (MDA) content in PeZFP38-OE lines was only about 0.7 times that of WT, indicating significantly reduced membrane lipid peroxidation damage (Figure 11G). Histochemical staining provided visual support for these data. The color intensity of DAB and NBT staining serves as an indicator of the accumulation levels of hydrogen peroxide (H2O2) and superoxide anion (O2-), respectively. No obvious difference in staining intensity was observed among untreated leaves (Figures 11H, I). However, under salt stress conditions, PeZFP38-OE plants exhibited lower accumulation of H2O2 and O2- compared to WT (Figures 11J, K). In summary, PeZFP38 overexpression enhances salt tolerance in Arabidopsis by promoting biomass accumulation, strengthening the antioxidant enzyme system, and reducing the accumulation of ROS under salt stress.
Figure 11. Overexpression of PeZFP38 in A. thaliana enhances tolerance to salt stress and improves ROS scavenging ability under hydroponic conditions. (A) Phenotypes of PeZFP38-OE transgenic lines and WT under normal conditions and 100mM NaCl treatment for 7 d. Bar = 1 cm. (B) Root length. (C) Fresh weight. (D) SOD activity. (E) CAT activity. (F) POD activity. (G) MDA content. (H) DAB staining. (I) NBT staining. (J) H2O2 content. (K) O2- content.
Earlier analysis revealed that the promoter of PeZFP38 was enriched with ABA-responsive elements (ABRE) and oxidative stress-associated elements (ARE). PeZFP38 was persistently induced in roots, stems, and leaves of P. euphratica under salt stress. Moreover, heterologous expression of PeZFP38 in Arabidopsis significantly enhanced the activities of antioxidant enzymes such as SOD and CAT. In summary, PeZFP38 likely functions as a transcriptional regulator that enhances salt tolerance by integrating ABA signaling with ROS scavenging systems, thereby activating downstream antioxidant and stress-adaptive genes, which collectively boost antioxidant capacity and mitigate oxidative damage.
4 Discussion
Zinc finger proteins constitute one of the largest families of TFs in plants, with C2H2-type zinc finger TFs being the most extensively studied. They play pivotal roles not only in regulating transcription during plant growth and development but also in responding to environmental stresses (Xie et al., 2019). To date, research on C2H2-ZFP gene family has predominantly focused on model plants such as A.thaliana (Lu et al., 2024) and tomato (T. Zhao et al., 2020). However, a systematic investigation encompassing genome-wide identification, expression profiling, and functional validation in the highly salt-tolerant tree species P. euphratica remains lacking.
In this study, we identified 67 PeZFPs, all of which contain zinc finger domains. This finding is consistent with previous reports on the C2H2-ZFP gene family (Pu et al., 2021). The number of PeZFPs is lower than that in its close relative P. trichocarpa (109 members) (Liu et al., 2015). This discrepancy may stem from different identification thresholds, but it could also indicate lineage-specific contraction and functional refinement of the gene family post-speciation. Based on protein similarity and evolutionary relationships, we classified the PeZFPs into five subfamilies. Similar subfamily classification results from other species (Liu et al., 2024; Zhou et al., 2024) support the robustness of this evolutionary framework. Notably, the expansion of the PeZFP gene family was driven by both tandem and segmental duplications, coupled with strong purifying selection. This evolutionary pattern, characterized by stable genomic duplication followed by selection pressure, is typical for stress-related gene families adapting to long-term environmental challenges (Singh and Sharma, 2024). It likely represents a key molecular underpinning for the adaptation of P. euphratica to its harsh, saline-alkaline riparian habitat. Furthermore, the promoter regions of PeZFPs were widely enriched with stress-responsive CREs such as ARE, ABRE, and MBS, reinforcing the hypothesis of an adaptive evolutionary shift toward a stress response function in this family.
Within this evolutionary framework, members exhibited significant structural and potential functional divergence. The vast majority PeZFPs contained the plant-specific QALGGH motifs, which is conserved in species like cucumber (Yin et al., 2020), Panax ginseng Meyer (Jiang et al., 2022), and Glycine soja (Liu et al., 2020). A total of 54 Q-type (80.6%) C2H2-ZFPs may function as transcriptional regulators broadly involved in stress responses. However, this assumption requires further experimental verification. For instance, many plant Q-type C2H2-ZFPs utilize EAR motifs to repress target genes, balancing growth and defense under stress (Xie et al., 2019). For example, PlZAT10 regulates the release of dormancy in peony seeds by binding to the promoter of the ABA catabolic gene PlCYP707A2 (Song et al., 2024). Importantly, under salt stress, most PeZFPs showed tissue-specific induction or repression patterns. These expression dynamics are closely linked to the specific combinations of hormone-related (e.g., ABA, jasmonate) and stress-responsive elements in their promoters, forming the basis of a refined transcriptional regulatory network for salt stress adaptation in P. euphratica. For example, some genes (e.g.,PeZFP3, PeZFP61) specifically highly induced in roots, whose promoters may be enriched with hypoxia (ARE) or root development-related elements, could play a role in the early perception and relay of salt stress signals in roots.
The findings indicate that most PeZFPs participate in pathways related to abiotic stress responses. Among them, PeZFP38 emerged as a key focus for functional analysis due to its multiple salient features. It belongs to subfamily IV, which contains P. euphratica-specific clades, hinting at possible functional innovation within this lineage. Its promoter is densely packed with hormone-related (e.g., ABRE for ABA response) and stress-responsive CREs. Under salt stress, PeZFP38 was rapidly and strongly induced in roots, stems, and leaves (with fold-changes up to 4.3–63.7), highlighting its important role in regulating response of poplar to salt stress. Heterologous overexpression experiments demonstrated that Arabidopsis lines overexpressing PeZFP38 significantly enhanced salt tolerance in, evidenced by increased biomass, elevated antioxidant enzyme (SOD, CAT) activities, and reduced oxidative damage (MDA). Based on these findings, we propose a mechanistic hypothesis for PeZFP38. Its role in enhancing antioxidant capacity suggests it may mitigate salt-induced oxidative damage by regulating key genes in the ROS scavenging pathway. A precedent exists in tree stress resistance. For instance, PeSTZ1 in P. euphratica was shown to enhance ROS scavenging and freezing tolerance by directly regulating the peroxidase gene PeAPX2 (He et al., 2020). In Betula platyphylla, SZA1 enhances salt tolerance by improving ROS scavenging capacity and proline accumulation, and is involved in the ABA/JA signaling pathways (Zhang et al., 2022b). Therefore, PeZFP38 likely functions as a TF that integrates upstream stress signals (potentially mediated by ABRE elements linked to the ABA pathway) to activate the transcription of downstream antioxidant and stress-protective genes, ultimately enhancing stress tolerance by maintaining ROS homeostasis (Bokolia et al., 2024). This framework logically connects the CRE features in its promoter, its strong induction pattern, and the observed physiological function of enhanced antioxidative defense.
Despite systematically identifying the PeZFP gene family and preliminarily revealing the function of PeZFP38, this study has limitations that outline future research directions. Our functional validation relied on a heterologous overexpression system. To conclusively demonstrate the necessity of PeZFP38 for salt tolerance in P. euphratica, subsequent studies should employ gene editing (e.g., CRISPR/Cas9-mediated knockout) or complementation tests in poplar. Future studies will utilize molecular biology techniques, such as ChIP-seq, to directly identify its downstream target genes, thereby mapping the complete antioxidant or ion homeostasis pathways it regulates. Although we predicted numerous CREs, the specific elements critical for driving the salt-induced expression of PeZFP38 require validation through promoter deletion or mutation analyses. This study did not investigate core salt tolerance parameters in P. euphratica, such as K+/Na+ homeostasis. Linking the function of PeZFP38 to these core ion homeostasis indicators would significantly enhance the physiological depth of the research.
5 Summary
In this study, we identified 67 C2H2-ZFP proteins in P. euphratica for the first time and classified them into five subfamilies, all containing zinc finger domains. Further evolutionary analysis indicated adaptive expansion of the family via duplication and purifying selection, establishing a preliminary framework for understanding functional divergence among its members. Phylogenetic analysis revealed that P. euphratica shares a closer evolutionary relationship with P. trichocarpa than with A. thaliana. Transcriptome analysis showed that most PeZFPs respond to salt stress and exhibit tissue-specific expression patterns, with PeZFP38 being significantly induced by salt stress. Both transient expression of PeZFP38 in poplar leaves and its stable overexpression in A. thaliana enhanced plant salt tolerance, which likely exerts its function by activating the ROS scavenging pathway. Collectively, this work represents the first systematic characterization of the C2H2-ZFP family in P. euphratica and demonstrates the key role of PeZFP38 in salt stress response. As a valuable candidate gene, PeZFP38 provides important genetic resources and a theoretical basis for elucidating the molecular mechanisms of salt tolerance in trees and promoting stress-resistant molecular breeding.
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Author contributions
YZ: Data curation, Investigation, Methodology, Writing – original draft. ZH: Conceptualization, Methodology, Supervision, Writing – original draft. LF: Data curation, Investigation, Writing – original draft. HL: Data curation, Investigation, Writing – original draft. HC: Data curation, Investigation, Writing – original draft. YY: Data curation, Investigation, Writing – original draft. NF: Data curation, Investigation, Writing – original draft. XH: Investigation, Methodology, Writing – review & editing. ZL: Conceptualization, Funding acquisition, Methodology, Writing – review & editing. RZ: Funding acquisition, Methodology, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Supported by the Fundamental Research Funds for the Central Non-profit Research Institution of Chinese Academy of Forestry (No. CAFYBB2025QF015), and the National Key Research and Development Program of China (No. 2021YFD2200201).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1754976/full#supplementary-material
Supplementary Table 1 | The physicochemical characteristics of PeZFP proteins in P. euphratica.
Supplementary Table 2 | The type and motif sequences of PeZFP proteins in P. euphratica.
Supplementary Table 3 | Ka/Ks values of PeZFPs duplicated gene pairs in P. euphratica.
Supplementary Table 4 | The expression patterns of PeZFPs in various tissues and under salt stress.
Supplementary Table 5 | Primers used in this study.
Supplementary Table 6 | List of Abbreviations for Proper Nouns.
Supplementary Figure 1 | qRT–PCR for PeZFP38 expression in PeZFP38-overexpressing leaves in Populus.
Supplementary Figure 2 | PCR detection of Arabidopsis lines overexpressing PeZFP38.
Supplementary Figure 3 | qRT–PCR for Arabidopsis lines overexpressing PeZFP38.
References
Admas, T., Shu, J., Shalmani, A., Pan, R., and Zhang, W. (2025). Salt stress-responsive transcription factors provide insights to enhance barley improvement: a review. Planta 262, 56. doi: 10.1007/s00425-025-04760-8
Alam, I., Wu, X., and Ge, L. (2022). Comprehensive genomic survey, evolution, and expression analysis of GIF gene family during the development and metal ion stress responses in soybean. Plants (Basel) 11, 570. doi: 10.3390/plants11040570
Ali, I., Salah, K. B. H., Sher, H., Ali, H., Ullah, Z., Ali, A., et al. (2024). Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana. Braz. J. Biol. 84, e259326. doi: 10.1590/1519-6984.259326
Arrey-Salas, O., Caris-Maldonado, J. C., Hernández-Rojas, B., and Gonzalez, E. (2021). Comprehensive genome-wide exploration of C2H2 zinc finger family in Grapevine (Vitis vinifera L.): insights into the roles in the pollen development regulation. Genes (Basel) 12, 302. doi: 10.3390/genes12020302
Biłas, R., Szafran, K., Hnatuszko-Konka, K., and Kononowicz, A. K. (2016). Cis-regulatory elements used to control gene expression in plants. Plant Cell Tiss. Organ Cult. 127, 269–287. doi: 10.1007/s11240-016-1057-7
Bokolia, M., Kumar, A., and Singh, B. (2024). Plant tolerance to salinity stress: regulating transcription factors and their functional role in the cellular transcriptional network. Gene Rep. 34, 101873. doi: 10.1016/j.genrep.2023.101873
Chen, C., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y., et al. (2020). Tbtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13, 1194–1202. doi: 10.1016/j.molp.2020.06.009
Chen, S., Jia, Y., Yang, Y., Liu, H., Chen, H., Liu, J., et al. (2025). Genome-wide analysis of the TsBLH gene family reveals TsBLH4 involved the regulation of abiotic stresses by interacting with KNOX6 in Toona sinensis. Plant Stress 15, 100721. doi: 10.1016/j.stress.2024.100721
Chen, Y. and Xia, P. (2025). NAC transcription factors as biological macromolecules responded to abiotic stress: a comprehensive review. Int. J. Biol. Macromol. 308, 142400. doi: 10.1016/j.ijbiomac.2025.142400
Deng, S., Wu, Y., Zeng, Q., Zhang, A., Duan, M., and Deng, M. (2024). Effects of Cd stress on morphological and physiological characteristics of maize seedlings. Agron. 14, 379. doi: 10.3390/agronomy14020379
Filiz, E. and Kurt, F. (2021). Expression and co-expression analyses of WRKY, MYB, bHLH and bZIP transcription factor genes in potato (Solanum tuberosum) under abiotic stress conditions: RNA-seq data analysis. Potato Res. 64, 721–741. doi: 10.1007/s11540-021-09502-3
Ge, X. L., Zhang, L., Du, J. J., Wen, S. S., Qu, G. Z., and Hu, J. J. (2022). Transcriptome analysis of Populus euphratica under salt treatment and PeERF1 gene enhances salt tolerance in transgenic Populus alba x Populus glandulosa. Int. J. Mol.Sci. 23, 3727. doi: 10.3390/ijms23073727
Han, Y. C. and Fu, C. C. (2019). Cold-inducible MaC2H2s are associated with cold stress response of banana fruit via regulating MaICE1. Plant Cell Rep. 38, 673–680. doi: 10.1007/s00299-019-02399-w
Han, G., Lu, C., Guo, J., Qiao, Z., Sui, N., Qiu, N., et al. (2020). C2H2 zinc finger proteins: master regulators of abiotic stress responses in plants. Front. Plant Sci. 11. doi: 10.3389/fpls.2020.00115
Han, G., Yuan, F., Guo, J., Zhang, Y., Sui, N., and Wang, B. (2019). AtSIZ1 improves salt tolerance by maintaining ionic homeostasis and osmotic balance in Arabidopsis. Plant Sci. 285, 55–67. doi: 10.1016/j.plantsci.2019.05.002
Hao, S., Wang, Y., Yan, Y., Liu, Y., Wang, J., and Chen, S. (2021). A review on plant responses to salt stress and their mechanisms of salt resistance. Hortic. 7, 132. doi: 10.3390/horticulturae7060132
He, F., Li, H. G., Wang, J. J., Su, Y., Wang, H. L., Feng, C. H., et al. (2019). PeSTZ1, a C2H2-type zinc finger transcription factor from populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2. Plant Biotechnol. J. 17, 2169–2183. doi: 10.1111/pbi.13130
He, F., Niu, M., Feng, C., Li, H., Su, Y., Su, W.-L., et al. (2020). PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus. Tree Physiol. 40, 1292–1311. doi: 10.1093/treephys/tpaa050
Huang, R., Jiang, S., Dai, M., Shi, H., Zhu, H., and Guo, Z. (2024). Zinc finger transcription factor MtZPT2–2 negatively regulates salt tolerance in Medicago truncatula. Plant Physiol. 194, 564–577. doi: 10.1093/plphys/kiad527
Huang, Y., Zhang, L., Zhang, K., Chen, S., Hu, J., and Cheng, F. (2022). The impact of tandem duplication on gene evolution in Solanaceae species. J. Integr. Agric. 21, 1004–1014. doi: 10.1016/s2095-3119(21)63698-5
Jia, H., Liu, G., Li, J., Zhang, J., Sun, P., Zhao, S., et al. (2020). Genome resequencing reveals demographic history and genetic architecture of seed salinity tolerance in Populus euphratica. J. Exp. Bot. 71, 4308–4320. doi: 10.1093/jxb/eraa172
Jiang, Y., Liu, L., Pan, Z., Zhao, M., Zhu, L., Han, Y., et al. (2022). Genome-wide analysis of the C2H2 zinc finger protein gene family and its response to salt stress in ginseng, Panax ginseng Meyer. Sci. Rep. 12, 10165. doi: 10.1038/s41598-022-14357-w
Lei, X., Fang, J., Zhang, Z., Li, Z., Xu, Y., Xie, Q., et al. (2024). PdbCRF5 overexpression negatively regulates salt tolerance by downregulating PdbbZIP61 to mediate reactive oxygen species scavenging and ABA synthesis in Populus davidiana × P. bolleana. Plant Cell Environ. 48, 1088–1106. doi: 10.1111/pce.15199
Li, B. and Dewey, C. N. (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf. 12, 323. doi: 10.1186/1471-2105-12-323
Li, D., Si, J., and Ren, X. (2024). Coordination and adaptation of water processes in Populus euphratica in response to salinity. Front. Plant Sci. 15. doi: 10.3389/fpls.2024.1443444
Lin, T., Yang, W., Lu, W., Wang, Y., and Qi, X. (2017). Transcription factors PvERF15 and PvMTF-1 form a cadmium stress transcriptional pathway. Plant Physiol. 173, 1565–1573. doi: 10.1104/pp.16.01729
Liu, Y., Khan, A. R., and Gan, Y. (2022). C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol.Sci. 23, 2730. doi: 10.3390/ijms23052730
Liu, H., Liu, Y., Liu, F., Zeng, L., Xu, Y., Jin, Q., et al. (2024). Genome-wide identification of the Q-type C2H2 zinc finger protein gene family and expression analysis under abiotic stress in lotus (Nelumbo nucifera G.). BMC Genomics 25, 648. doi: 10.1186/s12864-024-10546-1
Liu, Y. T., Shi, Q. H., Cao, H. J., Ma, Q. B., Nian, H., and Zhang, X. X. (2020). Heterologous expression of a Glycine soja C2H2 zinc finger gene improves aluminum tolerance in Arabidopsis. Int. J. Mol.Sci. 21, 2754. doi: 10.3390/ijms21082754
Liu, Q., Wang, Z., Xu, X., Zhang, H., and Li, C. (2015). Genome-wide analysis of C2H2 zinc-finger family transcription factors and their responses to abiotic stresses in poplar (Populus trichocarpa). PloS One 10, e0134753. doi: 10.1371/journal.pone.0134753
Lu, Y., Wang, K., Ngea, G. L. N., Godana, E. A., Ackah, M., Dhanasekaran, S., et al. (2024). Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses. J. Exp. Bot. 75, 5501–5520. doi: 10.1093/jxb/erae278
Ma, L., Han, R., Yang, Y., Liu, X., Li, H., Zhao, X., et al. (2023). Phytochromes enhance SOS2-mediated PIF1 and PIF3 phosphorylation and degradation to promote Arabidopsis salt tolerance. Plant Cell 35, 2997–3020. doi: 10.1093/plcell/koad117
Ndayambaza, B., Si, J., Zhao, X., Zhao, Y., Zhou, D., Jia, B., et al. (2025). Comprehensive genomic analysis of Trihelix transcription factor genes and their expression underlying abiotic stress in Euphrates poplar (Populus euphratica). Plants (Basel) 14, 662. doi: 10.3390/plants14050662
Nikraftar, S., Ebrahimzadegan, R., Majdi, M., and Mirzaghaderi, G. (2024). Genome-wide analysis of the C2H2-ZFP gene family in Stevia rebaudiana reveals involvement in abiotic stress response. Sci. Rep. 14, 6164. doi: 10.1038/s41598-024-56624-y
Niu, M., Feng, C., Liu, M., Liu, X., Liu, S., Liu, C., et al. (2024). Genome-wide identification of poplar GSTU gene family and its PtrGSTU23 and PtrGSTU40 to improve salt tolerance in poplar. Ind. Crops Prod. 209, 117945. doi: 10.1016/j.indcrop.2023.117945
Polle, A. and Rennenberg, H. (2019). Physiological responses to abiotic and biotic stress in forest trees. Forests 10, 711. doi: 10.3390/f10090711
Pu, J., Li, M., Mao, P., Zhou, Q., Liu, W., and Liu, Z. (2021). Genome-wide identification of the Q-type C2H2 transcription factor family in Alfalfa (Medicago sativa) and expression analysis under different abiotic stresses. Genes 12, 1906. doi: 10.3390/genes12121906
Qu, M., Sun, Q., Chen, N., Chen, Z., Zhang, H., Lv, F., et al. (2023). Functional characterization of a new salt stress response gene, PeCBL4, in Populus euphratica Oliv. Forests 14, 1504. doi: 10.3390/f14071504
Shao, L., Li, L., Huang, X., Fu, Y., Yang, D., Li, C., et al. (2023). Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi. BMC Plant Biol. 23, 298. doi: 10.1186/s12870-023-04298-5
Singh, A. and Sharma, A. K. (2024). Comparative analysis of the WRKY gene family reveals the gene family expansion and evolution in diverse plant species. Trends Hortic. 7, 5483. doi: 10.24294/th.v7i2.5483
Song, W., Sun, T., Xin, R., Li, X., Zhao, Q., Guan, S., et al. (2024). PlZAT10 binds to the ABA catabolism gene PlCYP707A2 promoter to mediate seed dormancy release in Paeonia lactiflora. Plant Cell Rep. 43, 276. doi: 10.1007/s00299-024-03363-z
Tyagi, K., V, P., Tyagi, P., Kumari, A., Pandey, R., Meena, N. L., et al. (2023). Seed priming with melatonin induces rhizogenesis and modulates physio-biochemical traits in high-yielding rice (Oryza sativa L.) genotypes. S. Afr. J. Bot. 163, 191–200. doi: 10.1016/j.sajb.2023.10.043
Wang, Z., Gerstein, M., and Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63. doi: 10.1038/nrg2484
Wang, Y., Liao, Y., Quan, C., Li, Y., Yang, S., Ma, C., et al. (2022b). C2H2-type zinc finger OsZFP15 accelerates seed germination and confers salinity and drought tolerance of rice seedling through ABA catabolism. Environ. Exp. Bot. 199, 104873. doi: 10.1016/j.envexpbot.2022.104873
Wang, X., Niu, Y., and Zheng, Y. (2021). Multiple functions of MYB transcription factors in abiotic stress responses. Int. J. Mol.Sci. 22, 6125. doi: 10.3390/ijms22116125
Wang, W., Wang, X., Wang, Y., Zhou, G., Wang, C., Hussain, S., et al. (2020). SlEAD1, an EAR motif-containing ABA down-regulated novel transcription repressor regulates ABA response in tomato. GM Crops Food 11, 275–289. doi: 10.1080/21645698.2020.1790287
Wang, D. R., Yang, K., Wang, X., and You, C. X. (2022a). A C2H2-type zinc finger transcription factor, MdZAT17, acts as a positive regulator in response to salt stress. J. Plant Physiol. 275, 153737. doi: 10.1016/j.jplph.2022.153737
Xie, M., Sun, J., Gong, D., and Kong, Y. (2019). The roles of Arabidopsis C1-2i subclass of C2H2-type zinc-finger transcription factors. Genes (Basel) 10, 653. doi: 10.3390/genes10090653
Yin, J., Wang, L., Zhao, J., Li, Y., Huang, R., Jiang, X., et al. (2020). Genome-wide characterization of the C2H2 zinc-finger genes in Cucumis sativus and functional analyses of four CsZFPs in response to stresses. BMC Plant Biol. 20, 359. doi: 10.1186/s12870-020-02575-1
Zhang, H., Sun, Z., Feng, S., Zhang, J., Zhang, F., Wang, W., et al. (2022a). The C2H2-type zinc finger protein PhZFP1 regulates cold stress tolerance by modulating galactinol synthesis in Petunia hybrida. J. Exp. Bot. 73, 6434–6448. doi: 10.1093/jxb/erac274
Zhang, X., Guo, Q., Qin, L., and Li, L. (2022b). A Cys2His2 zinc finger transcription factor BpSZA1 positively modulates salt stress in Betula platyphylla. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.823547
Zhao, C., Zhang, H., Song, C., Zhu, J. K., and Shabala, S. (2020a). Mechanisms of plant responses and adaptation to soil salinity. Innovation (Camb) 1, 100017. doi: 10.1016/j.xinn.2020.100017
Zhao, T., Wu, T., Zhang, J., Wang, Z., Pei, T., Yang, H., et al. (2020b). Genome-wide analyses of the genetic screening of C2H2-type zinc finger transcription factors and abiotic and biotic stress responses in tomato (Solanum lycopersicum) based on RNA-seq data. Front. Genet. 11. doi: 10.3389/fgene.2020.00540
Zhou, X., Gao, T., Zhang, Y., Han, M., Shen, Y., Su, Y., et al. (2024). Genome-wide identification, characterization and expression of C2H2 zinc finger gene family in Opisthopappus species under salt stress. BMC Genomics 25, 385. doi: 10.1186/s12864-024-10273-7
Keywords: C2H2-ZFP gene family, expression patterns, PeZFP38 gene, Populus euphratica, salt stress
Citation: Zhao Y, He Z, Fan L, Liu H, Chen H, Yang Y, Fan N, Han X, Lu Z and Zhuo R (2026) Genome-wide identification of the C2H2 zinc finger gene family in Populus euphratica and the functional analysis of PeZFP38 under salt stress. Front. Plant Sci. 17:1754976. doi: 10.3389/fpls.2026.1754976
Received: 26 November 2025; Accepted: 06 January 2026; Revised: 31 December 2025;
Published: 26 January 2026.
Edited by:
Shang Wang, UFZ-Helmholtz Centre for Environmental Research, GermanyReviewed by:
Hanzeng Wang, Jilin Agricultural Science and Technology College, ChinaYing Liu, Inner Mongolia University, China
Copyright © 2026 Zhao, He, Fan, Liu, Chen, Yang, Fan, Han, Lu and Zhuo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zhuchou Lu, bHV6Y0BjYWYuYWMuY24=; Renying Zhuo, emh1b3J5QGdtYWlsLmNvbQ==
†These authors have contributed equally to this work