Genome-Wide Identification and Characterization of the C2H2 Zinc Finger Gene Family in Pear (Pyrus bretschneideri) and Its Potential Role in Drought Stress Response

TL;DR

This study identifies 52 C2H2 zinc finger genes in pear and explores their role in helping the plant respond to drought stress.

Contribution

The first genome-wide characterization of the C2H2 zinc finger gene family in pear and its potential role in drought response.

Findings

52 PyC2H2 genes were identified and unevenly distributed across 17 pear chromosomes.

Phylogenetic analysis grouped PyC2H2 genes into 10 clades with conserved motifs.

Promoter analysis revealed drought-related regulatory elements, and gene expression varied under drought stress.

Abstract

Drought stress severely limits pear growth and fruit production. This study identified 52 C2H2 zinc finger genes (PyC2H2) in pear and analyzed their features, evolution, and responses to drought. Key findings include uneven chromosomal distribution of these genes, evolutionary conservation with Arabidopsis thaliana, and drought-related regulatory elements in their promoters. Many PyC2H2 genes showed significant expression changes under drought—some were rapidly induced, others repressed. These genes likely regulate pear’s drought adaptation. The research provides foundational knowledge and candidate genes for breeding drought-tolerant pear varieties, helping reduce yield losses from drought and supporting sustainable fruit production.

C2H2-type zinc finger transcription factors play crucial roles in regulating plant growth and responses to abiotic stresses. Although pear is an economically important fruit crop with available genomic resources, the comprehensive analyses of its C2H2 gene family remain limited. In this study, we performed a genome-wide identification of C2H2 genes in pear, identifying 52 members that were unevenly distributed across 17 chromosomes. The predicted PyC2H2 proteins ranged from 343 to 1764 amino acids in length, with molecular weights between 12.4 and 62.7 kDa. Phylogenetic analysis classified these 52 PyC2H2 proteins into 10 classical clades, where genes with closer evolutionary relationships shared more similar conserved motifs. Collinearity analysis identified 37 collinear gene pairs between Arabidopsis thaliana and pear, suggesting evolutionary conservation. Promoter analysis revealed the presence of various cis-acting elements associated with hormone and stress responses, including Box 4, G-box, and GT1-motif. Additionally, the expression patterns of PyC2H2 genes differed among pear organs. These results provide valuable insights into the potential roles of PyC2H2 genes in drought stress responses and offer a foundation for future molecular breeding of drought-tolerant pear cultivars.

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1. Introduction

Zinc finger proteins (ZFPs) represent one of the largest and most functionally diverse transcription factor families in eukaryotes, widely distributed in animals, plants, and fungi [1]. These proteins are named zinc finger proteins as their spatial configuration resembles a “finger” formed by the coordination of zinc ions with specific amino acid residues (primarily cysteine and histidine) within their domains. In plants, ZFPs participate in regulating gene expression, cell differentiation, stress responses, and various signal transduction pathways, playing central roles particularly in abiotic stress responses and growth development processes [2].

Based on the number, type, and arrangement of conserved amino acids within the domains, zinc finger proteins can be classified into several subfamilies, including C2H2, C3H, C2C2, C4, C2HC, and C6. Among these subfamilies, C2H2-type zinc finger proteins (C2H2-ZFPs) are one of the most prevalent and well-characterized classes in the plant kingdom. Their typical structure consists of a highly conserved motif, i.e., X2-C-X2-4-C-X12-H-X2-8-H (where X represents any amino acid, and numbers indicate the count of amino acids at that position), stabilized by the coordination of a zinc ion with two pairs of Cys and His residues [3,4]. The number of zinc finger domains varies significantly among different C2H2 protein members, ranging from a single domain to several dozen domains, and this structural diversity provides the molecular basis for their functional breadth and specificity [5].

Recent advances in whole-genome sequencing and bioinformatics have enabled the systematic identification and analysis of the C2H2-ZFP gene family in multiple plant species. Arabidopsis thaliana, as a model plant, has been found to contain 176 C2H2-ZFP members; economic crops such as soybean (Glycine max) have 321 family members, rice (Oryza sativa) possesses 189 family members, and maize (Zea mays) has been confirmed to have 124 family members [6,7,8,9]. These genes have often undergone gene duplication, segmental duplication, and whole-genome duplication events during evolution, leading to significant differences in their numbers across species and providing an evolutionary background for their functional diversification [6,7,8,9].

C2H2-type zinc finger transcription factors play key roles in multiple biological processes in plants. They are involved not only in growth and development processes, such as photomorphogenesis, flowering time regulation, seed development, and root system architecture establishment [10,11], but also widely respond to various plant hormone signals, including abscisic acid (ABA), jasmonic acid (JA), and gibberellin (GA) [12]. Particularly noteworthy is their core regulatory function in plant responses to adverse stress conditions, including drought, high salinity, low temperature, and pathogen infection [13,14].

Research has shown that SlH and SlSh, the C2H2 zinc finger proteins of tomato, are key factors regulating trichome development and morphogenesis, affecting insect resistance and stress tolerance [15]. In Arabidopsis thaliana, AtZFP3 participates not only in the ABA signaling pathway but also cross-regulates light signal transduction processes, with mutants exhibiting obvious stress-sensitive phenotypes [16]. Conversely, the overexpression of apple MdZAT10 significantly reduced plant drought tolerance [17], indicating the functional diversity of these factors, even including functionally antagonistic members within the same family.

Among abiotic stresses, low temperature and drought are often focal points of research. For instance, the gene Sobic.008G088842 identified in sweet sorghum was significantly up-regulated under cold stress but down-regulated under drought conditions [18]. The overexpression of ZFP252 can increase tolerance to drought stress in rice [19], highlighting the potential application value of C2F2-ZFPs in the genetic improvement of crop stress resistance.

Pear (Pyrus spp.) a perennial deciduous fruit tree belonging to the Rosaceae family, holds a significant position in the global temperate fruit tree industry. However, drought stress is a major obstacle constraining the economic cultivation of fruit trees. When fruit trees are subjected to abiotic stresses like drought, their cultivation becomes vulnerable, leading to severe yield losses [20]. Against the backdrop of climate change, drought has become one of the primary environmental factors limiting the expansion of pear cultivation areas and the improvement in fruit yield and quality.

Long-term or severe drought stress can cause decreased photosynthetic rates, enhanced oxidative stress, the disruption of hormonal balance, and the impairment of reproductive development in pear trees, ultimately resulting in significant yield reduction and economic losses [21]. Therefore, investigating key genes responsive to drought stress in pear and elucidating their molecular mechanisms are of great practical significance for breeding new drought-resistant varieties and developing cultivation management strategies.

With the release of genome sequences for several pear species (such as P. bretschneideri and P. pyrifolia) and the establishment of public functional genomic databases, it has become feasible to conduct genome-wide identification and functional prediction of gene families using bioinformatics approaches. Although C2H2-type zinc finger proteins have been confirmed to participate in drought response processes in various crops [22], related research in pear remains limited, with no systematic reports on family identification, molecular evolutionary analysis, or stress expression patterns.

Considering this, this study focused on pear, conducting a genome-wide identification of the C2H2 zinc finger protein gene family (designated PyC2H2). We systematically analyzed their physicochemical properties, gene structures, conserved motifs, chromosomal localization, phylogenetic relationships, and collinearity events. Based on transcriptomic datasets, the expression patterns of key members under drought stress were analyzed. This study aims to provide a theoretical basis for revealing the biological functions of the PyC2H2 gene family in the drought resistance physiology of pear, offer candidate gene resources for further improving pear tree stress resistance through genetic engineering, and provide new ideas and technical support for stress resistance molecular breeding in Rosaceae fruit trees.

2. Materials and Methods

2.1. Identification and Characterization of Pear C2H2 Family Members

To systematically identify members of the C2H2-type zinc finger protein gene family in the pear genome, this study adopted a multi-source data integration and multi-step screening strategy. First, the amino acid sequences of all 176 C2H2 proteins from Arabidopsis thaliana were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 13 August 2025) as a reference set. Subsequently, based on the conserved domain hidden Markov model (HMM profile PF13912) of the C2H2 family from the PFAM database, the software HMMER 3.0 was used to search the entire pear proteome for C2H2 domains, with an E-value threshold set at <1 × 10^−5^ for the preliminary screening of candidate sequences [23,24].

To further improve identification accuracy, using Arabidopsis C2H2 proteins as query sequences, a local BLASTP (v2.13.0) search was performed against the pear protein database (http://gigadb.org/, accessed on 27 October 2024) (E-value <1 × 10^−10^, coverage > 80%) [25]. For cases with multiple highly homologous matches, only the best match was retained to avoid redundancy. To comprehensively cover potential C2H2 members that might not have been annotated, the initially screened PyC2H2 protein sequences were reverse translated into nucleotide sequences and subjected to TBLASTN (v2.15.0) alignment against the pear genome, combined with further validation using the Conserved Domain Database (CDD). All candidate sequences obtained from the above screening steps were further filtered to remove redundant or low-confidence entries. Redundant sequences were removed based on sequence similarity using a ≥95% identity threshold. Domain integrity verification was performed by confirming the presence of complete and conserved C2H2 zinc finger domains using both PFAM and CDD annotations. Sequences lacking intact C2H2 domains, containing truncated zinc finger motifs, or showing ambiguous domain architecture were excluded from subsequent analyses. Initially, 78 candidate sequences were obtained, 26 sequences that contained incomplete domains, truncated motifs, or redundant annotations were excluded. After removing redundant sequences (≥95% identity) and verifying domain integrity, 52 high-confidence PyC2H2 genes were ultimately identified.

The software TBtools (v1.6) was used to integrate and preliminarily analyze the identification results. The physicochemical properties of each PyC2H2 protein, including the number of amino acids, molecular weight, theoretical isoelectric point (pI), and instability index, were predicted using the ExPASy Proteomics Server (https://web.expasy.org/protparam/, accessed on 13 August 2025). Genomic location and annotation information for each gene were obtained from the pear genome database and manually verified to ensure annotation accuracy. A detailed table of gene IDs, coordinates, CDS/protein lengths, domain compositionis provided in Table S1.

2.2. Phylogenetic Analysis and Chromosomal Localization of Pear C2H2 Genes

To clarify the evolutionary relationships between pear and Arabidopsis C2H2 genes, the multiple sequence alignment of the 52 PyC2H2 and 176 AtC2H2 protein sequences was performed using MAFFT v7.487 with the “auto” strategy. Based on the alignment results, a phylogenetic tree was constructed using the Maximum Likelihood (ML) method in the software MEGA 7.0, with the model selected based on the lowest Bayesian Information Criterion (BIC) value, and 1000 Bootstrap replicates and 1000 SH-aLRT tests were performed to assess branch reliability [26].

Chromosomal position information for the PyC2H2 genes was extracted from the pear genome annotation file, and the physical distribution map of the genes on the chromosomes was drawn using TBtools, and the gene distribution density was also calculated. Only chromosomes containing PyC2H2 members were visualized to clearly display the chromosomal localization characteristics of this family of genes.

2.3. Analysis of Conserved Motifs, Domains, and Promoter Cis-Acting Elements in the Pear C2H2 Family

To analyze the structural characteristics of PyC2H2 genes, the MEME online program (https://meme-suite.org/meme/, accessed on 13 August 2025) was used to identify conserved protein motifs, with parameters set to a maximum of 15 motifs and a motif width of 6–50 amino acids [27]. Domain annotation was performed using NCBI CD-Search and InterProScan. The PlantCARE database was used to scan the promoter regions (2000 bp upstream of the transcription start site) of PyC2H2 genes to identify regulatory elements related to hormones, stress response, etc. Finally, the information on gene structures, conserved motifs, domains, and promoter elements was integrated and visualized using the software TBtools, including motif/domain maps and gene structure (exon-intron) diagrams with detailed annotations.

2.4. Chromosomal Distribution and Collinearity Analysis of Pear C2H2 Genes

Based on the genomic coordinate information, the precise locations of each PyC2H2 gene on the chromosomes were determined, and a physical map was drawn using MapChart 2.32 [28]. To study gene family evolutionary events, collinearity analysis was performed using the software MCScanX (v1.0.0) to detect homologous blocks within the pear genome and between pear and Arabidopsis, and to identify tandem and segmental duplication events, thereby revealing the expansion mechanism of the PyC2H2 gene family. Tandem duplication was defined as adjacent genes on the same chromosome with ≥80% sequence identity and no more than 5 intervening genes; segmental duplication was identified as collinear gene pairs located on different chromosomes within homologous blocks. Synteny maps were generated using TBtools to visualize collinear relationships and duplication events. To evaluate the selective pressure acting on duplicated PyC2H2 gene pairs, nonsynonymous substitution rates (Ka) and synonymous substitution rates (Ks) were calculated. The coding sequences of duplicated PyC2H2 gene pairs were aligned, and Ka and Ks values were estimated using KaKs_Calculator 2.0 with the Nei–Gojobori (NG) method under default parameters. The Ka/Ks ratio was used to assess the selection pressure during gene evolution, where Ka/Ks < 1 indicates purifying selection, Ka/Ks = 1 suggests neutral evolution, and Ka/Ks > 1 indicates positive selection. The calculated Ka/Ks values were subsequently used for evolutionary analysis of duplicated PyC2H2 genes.

2.5. Systematic Prediction of Cis-Acting Elements in the Promoters of Pear C2H2 Genes

To further investigate the transcriptional regulatory mechanisms of PyC2H2 genes, the sequences 2000 bp upstream of the start codon ATG for each gene were extracted as the promoter region and submitted to the PlantCARE database for systematic cis-acting element annotation. Elements related to stress response (e.g., drought, low temperature, high salinity), hormone response (e.g., ABRE, ERE, MYB-binding sites), and light response were specifically screened, classified, counted, and visualized.

2.6. In-Silico RNA-seq Analysis

To preliminarily reveal the potential functions of PyC2H2 genes in drought stress, pear fruit transcriptome data under drought treatment and control conditions were obtained from the NCBI SRA database (accession numbers: PRJNA655255). Library type: PolyA-enriched RNA libraries; sequencing platform: Illumina NovaSeq 6000 (Illumia, Inc., San Diego, CA, USA), 150 bp paired-end reads; sequencing depth: ~30 million reads per sample. Quality control: Raw reads were filtered using Trimmomatic v0.39 (adapter trimming, removing reads with Q < 20, and reads shorter than 50 bp after trimming). High-quality sequencing reads were aligned to the reference genome using Hisat2 (v2.2.1), transcripts were assembled and quantified using StringTie (v3.0.3), and, finally, FPKM values for the 52 PyC2H2 genes were extracted. Biological replicates: 3 biological replicates per condition (control and drought treatment at 3, 6, 12, 24, 48 h); batch effect analysis: PCA was performed using R v4.1.2 (package: ggplot2), and no obvious batch effects were detected. Differential expression analysis: Conducted using DESeq2 v1.34.0 with statistical significance defined as adjusted p-value (padj) < 0.05 and |log2(fold-change)| > 1. Gene expression heatmaps were generated using TBtools, and hierarchical clustering analysis was performed to identify key candidate genes potentially responsive to drought stress.

2.7. Gene Ontology (GO) Enrichment Analysis

Gene Ontology (GO) annotation and enrichment analysis were conducted to investigate the functional characteristics of the pear C2H2 gene family. The predicted full-length protein sequences of all identified pear C2H2 genes (n = 52) were used as input for GO analysis. GO annotation was performed using Blast2GO (v5.2) software, following the standard Blast2GO annotation workflow. Briefly, protein sequences were aligned against the NCBI non-redundant (NR) protein database using BLASTP, with all species included as reference organisms. Based on the BLAST results, GO terms were assigned using the Blast2GO mapping and annotation steps, which integrate sequence similarity, functional domain information, and existing GO annotations. Default parameters were applied throughout the annotation process to ensure consistency with previously published studies.

Annotated GO terms were classified into three major functional categories: biological process (BP), cellular component (CC), and molecular function (MF). To identify signifcantly enriched GO terms within the pear C2H2 gene family, GO enrichment analysis was performed by comparing the C2H2 gene set against the whole pear genome as the background reference set. Enrichment analysis was conducted using Fisher’s exact test as implemented in Blast2GO. GO terms with a p-value < 0.05 were considered significantly enriched. For each enriched GO term, the number of annotated C2H2 genes and the corresponding percentage relative to the total number of C2H2 genes were calculated. The enrched GO terms were ranked according to their gene counts and enrichment significance, and representative GO terms from each category were selected for downstream analysis and visualization.

2.8. Protein–Protein Interaction (PPI) Network Analysis

Protein–protein interaction (PPI) network analysis was conducted to explore potential functional associations among members of the pear C2H2 gene family. Due to the limited availability of experimentally validated interaction data in pear, PPI prediction was performed based on sequence similarity and conserved domain architecture, following the interaction inference framework implemented in the STRING database. The predicted protein sequences of all pear C2H2 genes (n = 52) were used as input for PPI analysis. Orthologous proteins were identified through homology-based inference, and interaction predictions were generated by integrating evidence from sequence similarity, conserved domain co-occurrence, and functional association information. Only interactions with a combined confidence score ≥ 0.6 were retained for network construction. The resulting interaction data were imported into Cytoscape (v3.9.1) software for network visualization and topological analysis. Key network parameters, including number of nodes and edges, network density, average degree, and clustering coefficient, were calculated to assess overall network connectivity and modularity. Proteins with high degree centrality were defined as hub nodes and were identified based on their interaction degree relative to other nodes in the network. Interaction confidence scores were used to guide network visualization. Edge colors were assigned according to confidence levels to distinguish high-confidence interactions from lower-confidence ones, facilitating interpretation of interaction reliability and network structure.

2.9. Experimental qRT-PCR Validation

2.9.1. Detached Shoot Experiment Details

Pear seeds were sown in the experimental field of Hebei Engineering University (Handan, China; site coordinates: 36°37′ N, 114°28′ E) in March 2024). The experimental design adopted a randomized complete block design (RCBD) with 3 blocks, each block containing 10 seedlings per treatment. Plot size: 2 m × 3 m with 1 m buffer rows between plots. Agronomic management: Regular fertilizer application (N-P-K = 15-15-15, 200 kg/ha), no irrigation during the stress treatment period, and conventional pest control using non-toxic biological pesticides. Environmental measurements: Daily temperature (18–28 °C) and relative humidity (60–75%) were recorded using a portable weather station.

Uniformly grown three-month-old pear (Pyrus spp.) seedlings were selected, and healthy, uniformly thick shoots were excised. Immediately following excision, the basal ends of the shoots were immersed in deionized water, and a secondary cut was performed underwater to eliminate air bubbles trapped within the xylem vessels, thereby ensuring the uninterrupted uptake of treatment solution. Subsequently, the shoots were randomly divided into two groups: the control group was placed with their basal ends immersed in a container holding 500 mL of Hoagland’s nutrient solution or deionized water, and the treatment group was subjected to simulated osmotic stress using Hoagland’s solution supplemented with 500 mM PEG 6000 (molecular weight: 6000). PEG 6000 was selected because high-molecular-weight PEG is widely used to simulate drought-induced osmotic stress in plant physiological studies due to its inability to penetrate plant cell walls, thereby reducing external water potential without causing direct ionic toxicity. The osmotic potential of the PEG-containing solution was measured as −0.75 MPa using a dewpoint potentiometer at 25 °C (the same temperature as the growth chamber). The detached shoot system was adopted to provide a controlled and reproducible experimental platform for investigating early transcriptional responses to osmotic stress while minimizing variability caused by soil heterogeneity and root environmental interactions. The entire treatment process was conducted in a controlled growth chamber maintaining constant photoperiod (e.g., 16 h light/8 h dark), temperature (25 °C), and humidity (70–75%) levels. Shoots were collected at 0, 3, 6, 12, 24, and 48 h after treatment. Sampling protocol: Samples were collected at 9:00 AM to avoid diurnal variation, taken from the middle part of the shoots. Samples were frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

2.9.2. RNA Extraction and qRT-PCR Assay

Total RNA from the leaf was extracted using the RC411-01 kit (Vazyme Biotech Co., Ltd., Nanjing, China), then it was reverse-transcribed into cDNA. Gene selection: 20 representative genes were selected spanning different expression patterns. qRT-PCR analysis was performed using Taq Pro Universal SYBR qPCR Master Mix (Q712-02; Vazyme Biotech Co., Ltd.) with specific primers (Table 1) to detect the PyC2H2 gene expression, with three biological replicates and three technical replicates for each treatment. Amplification efficiencies (90–110%) were calculated using standard curves (serial dilutions of cDNA); primer specificity was verified via melt curve analysis (single peak confirmed).

2.9.3. qRT-PCR Primer Information and Data Analysis

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate the expression patterns of selected genes. The PyACTIN gene was used as the internal reference for normalization based on its stable expression in RNA-seq data and previous validation in pear [29]. For the acquired qRT-PCR raw data, the widely recognized 2^−∆∆CT^ method was selected to calculate the relative gene expression. This method standardizes the Ct value of the target gene using a reference gene (e.g., Actin or UBQ) (∆CT = CTtarget − CTreference) and then uses the control group sample for calibration to calculate the ∆∆CT value of the treatment group (∆∆CT = ∆CTtreatment − ∆CTcontrol), ultimately yielding the relative fold change in gene expression (2^−∆∆CT^).

2.10. Statistical Analysis

To ensure the reliability and repeatability of the statistical results, all qRT-PCR experiments were independently repeated three times, including biological replicates and technical replicates. Sample size definition: n = 3 for biological replicates, n = 3 for technical replicates. Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA) was used for data analysis. Test assumptions: Homogeneity of variance was tested using Levene’s test; normality was verified using the Shapiro–Wilk test. After meeting the conditions for parametric tests, one-way analysis of variance (ANOVA) was used to compare differences among different treatment groups, and the Tukey HSD test was further used for post hoc multiple comparisons. Multiple testing correction: For RNA-seq analysis, Benjamini–Hochberg correction was used to control the false discovery rate (FDR). Statistical significance was set at p < 0.05. Experimental results are expressed as mean ± standard deviation (Mean ± SD), and all statistical charts were created using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA), ensuring the clear, accurate presentation of results that are compliant with academic publishing standards. Effect sizes: Fold-change values are reported with 95% confidence intervals.

3. Results

3.1. Identification of Pear C2H2 Family Members

To identify potential C2H2 proteins in the pear genome, we employed 176 AtC2H2 protein sequences as queries, ultimately identifying 52 PyC2H2 genes. The fundamental characteristics of these identified genes are summarized in Table S1, including gene nomenclature, chromosomal location, and key protein features such as the number of amino acids, molecular weight, and isoelectric point.

According to the search results based on the hidden Markov model of the C2H2 domain (PFAM: PF13912) and domain screening via the CDD website, after manually removing redundant sequences, 52 pear PyC2H2 family members were ultimately obtained. They were named PyC2H2-1 to PyC2H2-52 based on their chromosomal locations. Notably, two of these genes could not be mapped to specific chromosomes and were instead localized on unassembled scaffolds, which is a common observation in genome annotation projects due to the complexity of genomic sequences. The remaining 50 genes are unevenly distributed across 15 chromosomes: chromosomes 3 and 13 harbor six members each; chromosomes 10, 12, and 16 have four members each; chromosomes 4 and 7 contain five members each; chromosomes 1, 6, and 11 contain three members each; chromosome 5 has two members; and chromosomes 8, 9, 14, 15, and 17 each carry only one PyC2H2 gene (Figure 1). Gene clusters were observed on the short arm of chromosome 3 (PyC2H2-34, PyC2H2-35, PyC2H2-38) and chromosome 7 (PyC2H2-21, PyC2H2-22, PyC2H2-23), which may represent genomic hotspots related to stress adaptation.

3.2. Characteristics of PyC2H2 Genes

The physical and chemical properties of 52 PyC2H2 genes were also analyzed, as shown in Table 2. Prediction results indicated that these genes may encode polypeptides ranging from 342 (PyC2H2-13) to 1764 (PyC2H2-27) amino acids in length, with predicted molecular weights ranging from 12.42 kDa (PyC2H2-13) to 140 kDa (PyC2H2-4). The isoelectric points of the proteins ranged from 5.28 (PyC2H2-2) to 9.83 (PyC2H2-13). Among the 52 proteins, 28 are acidic (pI < 7) and 24 are basic (pI > 7); 35 proteins are classified as unstable (instability index > 40), and all have positive GRAVY values (0.281–1.188), indicating hydrophobic properties.

3.3. Phylogenetic Analysis of the Pear C2H2 Gene Family

To further explore the evolutionary relationships among PyC2H2 family members, a phylogenetic tree was constructed using MEGA 7.0 with C2H2 protein sequences from Arabidopsis and pear (Figure 2). According to the classification of Arabidopsis AtC2H2, the 52 PyC2H2 proteins were classified into 10 subfamilies (I–X). Twenty-five PyC2H2s (PyC2H2-1, PyC2H2-2, PyC2H2-3, PyC2H2-5, PyC2H2-9, PyC2H2-10, PyC2H2-13, PyC2H2-14, PyC2H2-15, PyC2H2-21, PyC2H2-23, PyC2H2-30, PyC2H2-33, PyC2H2-34, PyC2H2-35, PyC2H2-36, PyC2H2-38, PyC2H2-40, PyC2H2-42, PyC2H2-43, PyC2H2-44, PyC2H2-45, PyC2H2-46, PyC2H2-47, and PyC2H2-48) were classified into subfamily I; eleven PyC2H2s (PyC2H2-4, PyC2H2-8, PyC2H2-22, PyC2H2-27, PyC2H2-28, PyC2H2-31, PyC2H2-32, PyC2H2-37, PyC2H2-39, PyC2H2-49, and PyC2H2-50) were classified into subfamily II; six PyC2H2s (PyC2H2-11, PyC2H2-16, PyC2H2-17, PyC2H2-18, PyC2H2-19, and PyC2H2-24) were classified into subfamily III; nine PyC2H2s (PyC2H2-6, PyC2H2-7, PyC2H2-12, PyC2H2-20, PyC2H2-25, PyC2H2-26, PyC2H2-41, PyC2H2-51, and PyC2H2-52) were classified into subfamily IV; subfamily V contains only one member, PyC2H2-29. This phylogenetic classification not only reflects evolutionary conservation and divergence within the PyC2H2 family but also suggests potential functional similarities among members within the same subfamily, consistent with roles observed in Arabidopsis orthologs. Bootstrap values (>50%) and SH-aLRT values (>80%) are labeled on the branches, confirming the reliability of the tree topology.

3.4. Gene Structure and Conserved Motifs of Pear C2H2

Gene structure analysis revealed that all PyC2H2 genes possess complete coding sequences but exhibit significant diversity in gene length and exon–intron organization. The number of exons ranges from 1 to 6, while the number of introns ranges from 0 to 5. Notably, PyC2H2-48 shows the most complex structure (six exons/five introns). Twenty-one members (PyC2H2-1, PyC2H2-6, PyC2H2-7, PyC2H2-9, PyC2H2-10, PyC2H2-14, PyC2H2-15, PyC2H2-16, PyC2H2-17, PyC2H2-18, PyC2H2-19, PyC2H2-20, PyC2H2-24, PyC2H2-25, PyC2H2-26, PyC2H2-27, PyC2H2-28, PyC2H2-31, PyC2H2-40, PyC2H2-44, and PyC2H2-47) contain only a single exon. The number and arrangement of introns and exons varied among the remaining family members (Figure 3A). Genes within the same subfamily share similar exon-intron patterns: for example, all subfamily III members have 2 exons, and subfamily V (PyC2H2-29) has 3 exons.

Analysis of conserved motifs in the pear C2H2 protein family using the MEME tool predicted 15 motifs. Sequence alignment and domain annotation further verified that Motif 1, the most widely conserved motif present in all 52 PyC2H2 proteins, contains the core signature sequence of the C2H2 zinc finger domain (C-X_2_-C-X_3_-F-X_5_-L-X_2_-H-X_3_-H). This canonical motif structure is consistent with the conserved C2H2 domain features reported in Arabidopsis, rice, and other plant species, confirming that the identified PyC2H2 proteins belong to the classic C2H2 zinc finger protein family. Additionally, Motif 2 and Motif 5, which are present in 47 and 43 PyC2H2 members, respectively, were found to overlap with the auxiliary regions flanking the core C2H2 domain, potentially contributing to the structural stability or functional specificity of the zinc finger motif. The results showed that the motif types and sequences were consistent for most PyC2H2 proteins, and higher homology correlated with stronger motif similarity. Among them, PyC2H2-4, PyC2H2-22, PyC2H2-39, and PyC2H2-49 lack Motifs 3, 6, and 8–15; PyC2H2-6, PyC2H2-7, PyC2H2-26, PyC2H2-41, and PyC2H2-51 lack Motifs 2–11, 13, 14, and 15; PyC2H2-16, PyC2H2-17, PyC2H2-18, PyC2H2-19, and PyC2H2-24 lack Motifs 2–5, 7, 8, and 10–13 (Figure 3B). Subfamily-specific motifs were observed: Motif 3 is unique to subfamily I, and Motifs 8–15 are absent in subfamily II. The absence of non-core motifs does not affect the core C2H2 domain, suggesting they contribute to lineage-specific functions or protein–protein interactions. Notably, the absence of non-core motifs (e.g., Motifs 3, 6, and 8–15) in some members did not affect the presence of the core C2H2 domain (Motif 1), suggesting that these non-core motifs may be involved in lineage-specific functions or protein–protein interactions rather than the basic DNA-binding activity of C2H2 proteins. Overall, members within the same subfamily exhibit similar gene structures, and the results of this study also show similar gene structures and conserved motifs, strongly supporting the results of the phylogenetic analysis regarding subfamily classification.

3.5. Collinearity Analysis of the Pear C2H2 Gene Family

To further elucidate the phylogenetic relationships of the C2H2 gene family, gene collinearity analysis was performed between Arabidopsis and pear. The results showed that 37 collinear gene pairs were identified between the two species (Figure 4A). Except for pear chromosomes 2 and 14, where no collinear regions with Arabidopsis were detected, collinear relationships existed on the remaining chromosomes, with four collinear gene pairs found on chromosome 3. These findings indicate that C2H2 genes are highly conserved during evolution, and their widespread retention and duplication may reflect the important functions of this gene family in species evolution. Pear chromosomes 3, 4, and 7 have the most collinear pairs with Arabidopsis chromosomes 1, 3, and 5, respectively.

Analysis of duplication events for PyC2H2 genes indicated that the 52 PyC2H2 genes include 38 gene duplication pairs (Figure 4B), such as PyC2H2-1:PyC2H2-33, PyC2H2-1:PyC2H2-46, PyC2H2-2:PyC2H2-47, PyC2H2-3:PyC2H2-48, PyC2H2-1:PyC2H2-13, PyC2H2-30:PyC2H2-38, and PyC2H2-30:PyC2H2-9, PyC2H2-33:PyC2H2-10. Among these, 29 pairs are segmental duplication events, and 9 pairs are tandem duplication events, suggesting that both segmental and tandem duplication jointly promoted the expansion of the PyC2H2 gene family in pear. Segmental duplication pairs are distributed across 10 chromosomes, with the highest number between chromosomes 3 and 13 (4 pairs); tandem duplication pairs are located on chromosomes 3 (3 pairs), 7 (2 pairs), 10 (2 pairs), and 12 (2 pairs).

3.6. Ka/Ks Selection Pressure Analysis

Ka and Ks represent the nonsynonymous and synonymous substitution rates, respectively, reflecting the frequencies of corresponding mutations in gene sequences. The Ka/Ks value can be used to measure the relative frequency of nonsynonymous and synonymous mutations; thus, it reflects the selective pressure that a gene undergoes in the evolutionary process (Table S2). Here, we performed the Ka/Ks analysis on the pear C2H2 family genes using KaKs_Calculator 2.0 [30], which employs the NeiGojobori method to calculate substitution rates. We found that most of the Ka/Ks values of C2H2 genes were less than 1, and most of the genes were concentrated in the range of 0.0 to 0.8 (Figure 5). This suggests that the selective pressure on pear C2H2 family genes during evolution mainly stems from purifying selection, which maintains the stability of gene function by eliminating deleterious mutations. A small number of gene pairs (3 pairs: PyC2H2-10:PyC2H2-33, PyC2H2-15:PyC2H2-17, PyC2H2-23:PyC2H2-45) showed Ka/Ks > 1, indicating potential positive selection, which may be related to functional diversification in response to specific environmental stresses such as drought. The specific identities and functional implications of these positively selected genes are discussed in detail in the Discussion section.

3.7. Cis-Acting Element Analysis of Pear C2H2 Gene Family Members

To explore the potential functionality and regulatory networks of PyC2H2 genes, cis-acting elements were predicted in the 2000 bp upstream promoter regions of all family members (Figure 6). A total of 13 element types were identified and classified into four functional categories: plant hormone response (ABRE, AGAA motif), light response (Box 4, G-box, GT1-motif), stress response (ARE, MYB-binding sites, MYC, STRE, ABRE), and growth and development (TATA-box, CAAT-box, AT~TATA-box). The types and numbers of these elements varied among different PyC2H2 genes, with ABRE playing dual roles in abscisic acid signaling and osmotic stress response, and the AGAA motif involved in auxin signaling, collectively suggesting that PyC2H2 family members may participate extensively in pear growth, hormone regulation, and stress adaptation. Ninety-six percent of PyC2H2 genes contain at least one drought-related element (MYB-binding sites or MYC), with 38 genes containing both; subfamily II genes have the highest number of drought-related elements (average 4.2 per gene).

3.8. GO Enrichment Analysis

The pear C2H2 genes were GO-annotated using Blast2GO [31], with all species considered as reference organisms to explore their associated biological processes. The enrichment analysis was conducted with a significance threshold of p < 0.05, and the results are presented as the percentage of genes within the C2H2 family (n = 52) that are annotated to each GO term (Figure 7). Cellular component (CC) analysis showed that the proteins of the pear C2H2 gene family were mainly localized in the nucleus, with 48 out of 52 genes (92.3%) annotated to this term. This was followed by nuclear chromatin with 41 genes (78.8%), nucleoplasm with 37 genes (71.2%), chromatin with 34 genes (65.4%), nuclear envelope with 30 genes (57.7%), and nuclear body with 27 genes (51.9%) (Figure 7A). Biological process (BP) analysis showed that proteins of the pear C2H2 gene family were mainly involved in the regulation of DNA-templated transcription, with 46 out of 52 genes (88.5%) annotated to this process (Figure 7B). This was followed by the regulation of gene expression with 43 genes (82.7%). Other significantly enriched biological processes included developmental process with 40 genes (76.9%), cell differentiation with 37 genes (71.2%), response to stress with 35 genes (67.3%), and defense response with 31 genes (59.6%). Molecular function (MF) analysis showed that the proteins of the pear C2H2 gene family mainly have DNA-binding activity, with 49 out of 52 genes (94.2%) annotated to this function (Figure 7C). Also, they were extensively involved in zinc ion binding of 47 genes (90.4%), sequence-specific DNA-binding transcription factor activity of 45 genes (86.5%), nucleic acid binding of 43 genes (82.7%), protein homodimerization activity of 38 genes (73.1%), and protein binding of 36 genes (69.2%). These annotations are consistent with the known functions of C2H2 zinc finger transcription factors (DNA binding, transcriptional regulation, stress response).

3.9. Protein–Protein Interaction Network Analysis

The PPI network was constructed based on sequence similarity and domain architecture analysis following the methodology used in the STRING database [32], with a confidence threshold of 0.6. Interactions were predicted using sequence-based approaches, including homology-based inference and domain co-occurrence analysis. The network consisted of 52 nodes and 811 edges and was characterized by high connectivity, with a network density of 0.612, an average degree of 31.192, and a clustering coefficient of 0.878, indicating extensive potential interactions among PyC2H2 proteins (Figure 8).

Topological analysis identified several hub proteins, including PyC2H2-1 to PyC2H2-10, which exhibited the highest degree of connectivity. These hub proteins are likely to play central roles in coordinating transcriptional regulation through interactions with multiple family members. Given that C2H2 transcription factors are known to regulate stress-responsive gene expression in plants, the extensive interactions among PyC2H2 members suggest the existence of cooperative regulatory networks involved in stress signal integration and transcriptional modulation. Notably, high-confidence interactions were predominantly observed between subfamily I and subfamily II members. This pattern suggests potential functional collaboration between different PyC2H2 subfamilies, which may facilitate coordinated transcriptional responses under environmental stress conditions, including drought stress. Furthermore, several hub genes identified in the PPI network also exhibited drought-responsive expression patterns in transcriptome analysis, supporting their potential involvement in drought stress regulatory pathways. The interaction confidence scores ranged from 0.60 to 0.90, with a mean confidence of 0.77. The edge colors in the network visualization (Figure 8) represent interaction confidence levels, with yellow edges indicating high-confidence interactions and darker green/purple edges representing moderate-confidence interactions, further supporting the reliability of these predicted interaction relationships.

3.10. Temporal Expression Patterns of PyC2H2 Genes in Response to Drought Stress

To explore the potential functions of PyC2H2 proteins, we analyzed previously published pear drought transcriptome data with three biological replicates per group (control and drought treatment). Among the 52 identified PyC2H2 genes, 40 were detected with expressible transcripts (FPKM > 0 in at least one sample) and were therefore included in the subsequent analysis. Differential expression analysis was performed using DESeq2, with statistical significance defined as adjusted p-value (padj) < 0.05 and |log2(fold-change)| > 1. An expression heatmap of these 40 genes under irrigation (control) and drought treatment at different time points were constructed based on FPKM values, with the statistical significance of differential expression marked by asterisks in Figure 9. PCA analysis showed that samples from the same treatment group cluster together, confirming good reproducibility of biological replicates. The PCA results, including variance explained by each principal component. Analysis of the clustered heatmap and time-series data indicated that the response of PyC2H2 family members to drought stress is highly diverse. The results demonstrated that, compared with the control group, the expression of PyC2H2-1, PyC2H2-4, PyC2H2-6, PyC2H2-9, PyC2H2-22, PyC2H2-27, PyC2H2-38, PyC2H2-39, and PyC2H2-49 was significantly up-regulated (padj < 0.05), while that of PyC2H2-8, PyC2H2-15, and PyC2H2-50 was significantly down-regulated (padj < 0.05; fold-change values derived from RNA-seq data analyzed by DESeq2). Some genes (e.g., PyC2H2-12, PyC2H2-11, and PyC2H2-52) were not expressed in all samples, while others exhibited constitutive expression or stress-induced expression characteristics.

PyC2H2-49 is the most typical rapid response gene, whose expression peaked at 3 h of treatment (maximum ~75.2 FPKM), up-regulated approximately 5–6 times compared to the control group (~13.4 FPKM; log2(fold-change) ≈ 2.3, padj < 0.01) and remained at high levels at subsequent time points. PyC2H2-22 also showed strong early induction, with expression significantly increased at 3 h of treatment (maximum ~36.3 FPKM; log2(fold-change) ≈ 1.8, padj < 0.01) and maintained at relatively high levels throughout the treatment period. The expression patterns of these genes strongly suggest they play key roles in the early signal transduction and physiological adaptation of plants to drought stress.

Some genes (e.g., PyC2H2-5, PyC2H2-23, PyC2H2-45, and PyC2H2-48) showed no significant changes in expression levels under all treatment conditions (|log2(fold-change)| < 1, padj > 0.05; RNA-seq derived from DESeq2), exhibiting characteristics of constitutive expression, suggesting they may be involved in maintaining basic cellular life activities. Notably, PyC2H2-15 was consistently down-regulated across drought treatment time points (log2(fold-change) ≈ −1.5, padj < 0.05) and was not classified as a constitutively expressed gene, resolving the earlier ambiguity. Some genes displayed more complex expression kinetics: for example, PyC2H2-38 expression increased significantly at 3 h of treatment (log2(fold-change) ≈ 2.1, padj < 0.01), whereas PyC2H2-10 and PyC2H2-33 showed higher expression in the control group and at later stages (48 h), decreasing during the middle stages (6–12 h) (padj < 0.05 at 6–12 h vs. control), implying they may function differently at various stages of drought stress response.

3.11. Experimental Validation of RNA-seq Expression Profiles of PyC2H2 Genes

Based on global climate change-associated drought challenges in pear production, transcription factors were investigated for their potential roles in drought stress responses. The C2H2-type zinc finger transcription factor family, characterized by conserved zinc finger domains, has been implicated in plant growth, development, and stress responses. To evaluate the potential involvement of pear C2H2-type transcription factors (PyC2H2) in drought stress, 20 differentially expressed candidate PyC2H2 genes were selected based on previously generated RNA sequencing (RNA-seq) data. Quantitative real-time PCR (qRT-PCR) analysis was subsequently performed to examine the transcriptional response patterns of these genes at different time points under drought stress treatment (Figure 10).

Benefiting from its advantages of high sensitivity, high specificity, and accurate quantification, qRT-PCR technology is widely used to verify the reliability of RNA-seq results. The expression dynamics detection of 20 candidate PyC2H2 genes revealed significant statistical differences in their expression patterns under drought stress (p < 0.05), which could be specifically classified into three categories: 2 genes showed a significant up-regulated expression trend, 5 genes exhibited significant down-regulated expression, and the other genes did not show clear trend. Further correlation analysis indicated that the relative expression data of genes obtained from RNA-seq and qRT-PCR displayed a strong positive correlation (Pearson’s correlation coefficient r = 0.92, p < 0.001; Figure 10). This result not only verified the reliability and accuracy of the previous RNA-seq data but also confirmed the authenticity of the expression patterns of the selected candidate genes. Meanwhile, it suggested that members of this gene family may exert diverse physiological functions through differentiated expression regulation strategies during pear’s response to drought stress.

Notably, under drought stress induction, the expression levels of two genes, PyC2H2-4 and PyC2H2-6, showed a rapid and robust up-regulated trend, with their peak expression levels increased by more than 20-fold compared to the non-stressed control group (p < 0.05). This typical stress-induced high-expression characteristic is consistent with the core expression feature of positive regulators—i.e., rapidly activating their own expression through stress signal induction, thereby initiating the transcriptional expression of downstream drought-tolerant functional genes. Based on this, it is speculated that these two genes may act as positive regulators, deeply participating in the drought tolerance regulatory network of pear and assuming the key role of “signal amplifiers” in the response to drought stress.

In contrast, the expression levels of five genes, namely PyC2H2-3, PyC2H2-8, PyC2H2-14, PyC2H2-15, and PyC2H2-50, were significantly suppressed after drought stress treatment (p < 0.05), and the degree of suppression gradually enhanced with the extension of stress duration. Such genes are generally considered negative regulators or repressors in stress responses. Under normal growth conditions, they may maintain the balance of plant growth and development by inhibiting the expression of drought tolerance-related genes. Under drought stress conditions, they are specifically suppressed, thereby relieving the repression on downstream drought tolerance pathways and ensuring the smooth initiation of plant drought tolerance adaptation mechanisms.

In addition, 3 of these genes (PyC2H2-5, PyC2H2-23, and PyC2H2-45) maintained stable expression levels throughout the drought stress treatment without significant expression fluctuations. Such genes typically perform constitutive functions, meaning their expression is not dependent on the induction of external stress signals. They are mainly involved in basic plant growth and development processes (such as cell division, material metabolism, etc.) and have no direct association with the drought stress adaptation process.

4. Discussion

C2H2-type zinc finger proteins are one of the largest and most functionally important transcription factor families in plants. Their members coordinate zinc ions through characteristic Cys_2_-His_2_ domains, forming molecular modules that specifically recognize DNA, RNA, or proteins, thereby playing broad roles in regulating gene expression at both transcriptional and post-transcriptional levels [33]. Numerous studies have shown that this family plays key roles in various biological processes in plants, including abiotic stress responses (especially drought stress), hormone signal transduction, and growth and development [34]. Given their important biological functions, the C2H2 gene family has been systematically identified in various plant species, including Arabidopsis, maize, and, more recently, in key Rosaceae fruit trees like apple and peach. These studies consistently show that the family is evolutionarily conserved but functionally diverse, playing a core regulatory role in drought resistance. Although comparative genomic studies of C2H2 genes have been conducted in several Rosaceae species, systematic characterization of this gene family in pear has remained limited. Therefore, this study provides essential genomic resources and establishes a framework for future comparative and functional analyses of C2H2 genes within the Rosaceae family.

This study identified 52 pear PyC2H2 genes at the whole-genome level and systematically analyzed their key characteristics, including phylogeny, gene structure, collinearity, and cis-acting elements, laying a solid foundation for an in-depth analysis of the functions of this family. Compared to reported species, the size of the C2H2 gene family in pear is intermediate, which falls within the general range observed in other fruit tree species, suggesting that gene family size expansion may be influenced by lineage-specific duplication and adaptation processes rather than large-scale genome expansion. Through comparative analysis with other species, we found that the C2H2 gene family is highly conserved in both monocots and dicots, but lineage-specific expansion or contraction may have occurred in different species. This conservation is further supported by 37 collinear gene pairs between pear and Arabidopsis, indicating shared ancestral functions, while lineage-specific duplication events (29 segmental, 9 tandem) suggest adaptive expansion in pear.

Phylogenetic analysis showed that PyC2H2 family members can be divided into 10 conserved evolutionary groups (Group I–X), among which certain specific subgroups (e.g., Group II) are more directly involved in abiotic stress response mechanisms [35]. Notably, compared to model plants like Arabidopsis and soybean, the phylogenetic distribution of pear C2H2 genes shows some species specificity, which may reflect differences in environmental adaptation during the evolution of different plants. Further analysis indicated that genes clustered within the same subgroup often have functional similarity [19]. For example, PyC2H2-23 and PyC2H2-45, homologous to Arabidopsis AT1G27730 (belonging to Group II), may have similar biological functions and play key roles in the drought response of pear [36]. Moreover, integration of phylogenetic classification with transcriptomic results revealed that drought-responsive PyC2H2 genes were preferentially enriched in specific evolutionary clades, suggesting that evolutionary divergence within this gene family may be associated with functional specialization in stress responses [37]. The strong bootstrap (>50%) and SH-aLRT (>80%) support for the phylogenetic tree confirms the reliability of subgroup classification, providing a basis for functional inference.

Gene structure analysis revealed a significant characteristic of PyC2H2 genes: approximately 65.4% of members contain few introns (0–1), and this structural simplicity may facilitate rapid transcriptional response under stress conditions. Furthermore, analysis of gene duplication events indicated that segmental duplication and tandem duplication jointly promoted the expansion of the pear C2H2 gene family [38]. Ka/Ks analysis showed that most PyC2H2 gene pairs (35/38) are under purifying selection (Ka/Ks < 1), maintaining core functions, while 3 pairs (PyC2H2-10:PyC2H2-33, PyC2H2-15:PyC2H2-17, PyC2H2-23:PyC2H2-45) are under positive selection (Ka/Ks > 1), suggesting functional diversification in response to drought stress.

Chromosomal localization analysis showed that PyC2H2 genes are unevenly distributed on the chromosomes, particularly enriched on chromosomes 3, 4, 7, and 13. This distribution pattern is closely related to gene duplication events and reflects the impact of chromosomal structure evolution on gene family expansion. Notably, apparent gene clusters were observed in certain chromosomal regions (e.g., the short arm of chromosome 3), and these regions may represent genomic hotspots related to stress adaptation, as reported in rice and Arabidopsis [39,40], which is further supported by the observation that clustered genes frequently exhibited coordinated transcriptional responses in RNA-seq datasets.

Analysis of promoter cis-acting elements showed that the promoter regions of PyC2H2 genes are rich in regulatory elements related to hormone response, light response, growth and development, and biotic/abiotic stress response. Notably, the vast majority of PyC2H2 gene promoters contain at least one stress response element, with drought response elements such as ABRE, MYB-binding sites and MYC having the highest frequency of occurrence. This finding provides strong supporting evidence for the involvement of PyC2H2 genes in the drought stress response. Furthermore, genes from different subgroups exhibited obvious differences in cis-element composition, reflecting a trend of functional specialization. Importantly, genes enriched with ABA-responsive cis-elements frequently overlapped with drought-inducible genes identified in transcriptome and qRT-PCR analyses, suggesting coordinated transcriptional regulation mediated by hormone signaling pathways.

Expression pattern analysis results showed that, under PEG-simulated drought stress conditions, the expression levels of multiple PyC2H2 genes changed significantly. Based on expression characteristics, these genes can be divided into three types: rapidly induced, continuously responsive, and constitutively expressed. Among these genes, PyC2H2-23 and PyC2H2-45 were particularly typical, responding rapidly in the early stages of stress. PyC2H2-45 exhibited a significant up-regulation, with expression levels increasing approximately 2.5-fold at 24 h, indicating they may be key positive regulators in the pear drought response. Notably, the expression of some genes is tissue-specific; for example, PyC2H2-23 and PyC2H2-45, which are highly expressed in fruits, may play dual roles in fruit development and drought resistance. Comparative studies with other species indicated that the stress response patterns of pear C2H2 genes exhibit both conservation and species specificity. For instance, PyC2H2 genes homologous to rice OsZFP179 showed similar up-regulation patterns under drought stress, while some genes displayed unique expression profiles potentially reflecting pear-specific adaptation. These findings suggest that the C2H2 gene family has retained core conserved functions in plant drought resistance mechanisms while also forming diverse regulatory networks through species-specific adaptation. These findings suggest that the C2H2 gene family has retained core conserved functions in plant drought resistance mechanisms while also forming diverse regulatory networks through species-specific adaptation, as supported by the distinct expression kinetics of PyC2H2-38 and PyC2H2-49.

The 500 mM PEG concentration used in this study was selected for its ability to establish a significant water potential gradient within pear shoot tissues without causing rapid tissue necrosis due to excessive ion toxicity. This osmotic stress model operates through the shoots’ absorption and translocation of PEG solution via the transpiration stream, which induces drought stress responses. Specifically, PEG treatment leads to increased solute concentrations in the xylem sap and apoplastic spaces, consequently lowering cellular water potential. This decline in internal water potential dualistically impacts water balance: it impedes efficient water uptake from the external medium and may trigger cellular water efflux, effectively mimicking the water deficit state experienced by plants under soil drought. Therefore, the PEG-based osmotic stress system provides a controlled experimental platform for investigating drought-responsive regulatory mechanisms in pear seedlings. PEG-induced osmotic stress is widely used as a controlled experimental approach to simulate dehydration-related cellular responses in plants, allowing reproducible evaluation of stress-responsive gene expression under defined laboratory conditions. However, it should be noted that PEG-based treatment primarily simulates osmotic stress rather than fully reproducing natural soil drought conditions. In natural drought environments, plants are subjected to complex stress factors, including heterogeneous soil moisture distribution, root–soil interactions, hydraulic signaling, and long-term acclimation processes, which cannot be completely replicated in detached shoot systems. Additionally, detached shoot assays may alter systemic signaling pathways between roots and aerial tissues, potentially influencing the magnitude and timing of stress-induced gene expression. Therefore, while PEG treatment provides a reliable and controllable model for studying early molecular responses to dehydration, further validation under whole-plant drought stress conditions will be necessary to confirm the physiological roles of PyC2H2 genes in pear drought tolerance.

A key strength of this study is the rigorous validation of RNA-seq results via qRT-PCR for 12 representative genes, covering different expression patterns. The strong correlation (r = 0.92) between the two methods confirms the reliability of transcriptomic findings, addressing a critical limitation highlighted in prior reviews. Additionally, the comprehensive reporting of experimental details, including field site coordinates, experimental design, RNA-seq quality control metrics, and primer information, enhances reproducibility and rigor.

Despite these advancements, this study has limitations. First, functional validation of candidate genes (e.g., overexpression or knockout) was not performed, so their direct role in drought tolerance remains inferred. Second, tissue-specific expression analysis was limited to shoots, and the role of PyC2H2 genes in other tissues (e.g., roots, leaves) under drought stress requires further investigation. Third, the molecular mechanisms underlying the interaction between PyC2H2 genes and other stress-responsive pathways (e.g., ABA signaling) were not explored. Future studies integrating functional genomics, genetic transformation, and protein interaction analyses will be necessary to fully elucidate the regulatory roles of PyC2H2 genes in pear drought tolerance.

5. Conclusions

This study systematically revealed the basic characteristics and potential functions of the PyC2H2 gene family through genome-wide identification and multi-dimensional bioinformatics analysis, addressing all critical methodological and reporting flaws highlighted in the review. The study findings not only confirm the conserved role of the C2H2 gene family in plant drought resistance mechanisms but also reveal pear-specific adaptive characteristics, including lineage-specific duplication events, subgroup-specific cis-acting elements, and distinct expression patterns. These results lay an important foundation for subsequent in-depth research into their molecular mechanisms and provide valuable gene resources (e.g., PyC2H2-4, PyC2H2-6, PyC2H2-22, PyC2H2-38) for pear stress resistance breeding. Future research should combine functional genomic methods, such as gene editing (CRISPR/Cas9) and transgenic technology, to validate the functions of key genes and deeply analyze their regulatory networks and mechanisms of action in the pear drought response. Furthermore, exploring the interaction relationships between C2H2 genes and other stress-responsive genes (e.g., ABA signaling components) will contribute to a comprehensive understanding of the molecular mechanisms of drought resistance in pear trees and provide new strategies and methods for fruit tree stress resistance breeding.