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Article

Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica)

by
Qian Zhao
1,
Yingxin Zhang
1,
Xiangyu Xing
1,
Shuyao Li
1,
Ruidong Sun
1,
Weilong Zhang
2,
Jun Zhang
1,
Liangyu Jiang
1,
Zhenyuan Zang
1,*,
Ming Gao
2,* and
Jian Zhang
1,3,*
1
Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, China
2
Northeast Innovation Center for Agricultural Science and Technology, Jilin Academy of Agricultural Sciences, Changchun 136100, China
3
Department of Biology, University of British Columbia Okanagan, Kelowna, BC V1V 1V7, Canada
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(7), 1618; https://doi.org/10.3390/agronomy15071618
Submission received: 10 May 2025 / Revised: 28 June 2025 / Accepted: 30 June 2025 / Published: 2 July 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

C2H2 zinc finger proteins (C2H2-ZFPs) constitute one of the largest transcription factor families in plants, playing crucial roles in growth, development, and stress responses. Here, we performed a comprehensive genome-wide analysis of C2H2-ZFPs in foxtail millet (Setaria italica v2.0), identifying 67 members that were unevenly distributed across all nine chromosomes. Most SiC2H2 proteins were predicted to be alkaline, stable, and nuclear-localized, with the exception of SiC2H2-11 and SiC2H2-66, which were chloroplast-targeted. Phylogenetic analysis with Arabidopsis thaliana and Oryza sativa (rice) homologs classified these genes into seven distinct subfamilies, each containing the characteristic motif1 domain. Evolutionary studies revealed 14 segmental duplication events and strong syntenic conservation with Triticum aestivum (wheat, 163 orthologous pairs), suggesting conserved functions during evolution. Promoter analysis identified multiple cis-acting elements associated with light responsiveness, hormone signaling, and stress adaptation. Transcriptome profiling and qPCR validation in the YuGu 56 cultivar identified several stress-responsive candidates, including SiC2H2-35 and SiC2H2-58 (salt tolerance), as well as SiC2H2-23 (5.19-fold induction under salt stress) and SiC2H2-32 (5.47-fold induction under drought). This study provides some valuable insights into the C2H2-ZFP family in foxtail millet and highlights potential genetic markers for improving stress resilience through molecular breeding approaches.

1. Introduction

Zinc finger proteins (ZFPs) are a major class of eukaryotic transcription factors, with C2H2-type zinc finger proteins (C2H2-ZFPs) being the most abundant and functionally important subtype [1]. These proteins contain a conserved structural domain of about 20–30 amino acids, which coordinates zinc ions (Zn2+) to form stable structures essential for their function [2]. Based on the arrangement of cysteine (Cys) and histidine (His) residues, ZFPs are classified into multiple subtypes, among which C2H2-ZFPs have been extensively studied in plants [3,4,5,6,7,8,9].
C2H2-ZFPs play critical roles in regulating plant growth, development, and response to abiotic stresses [10]. In Arabidopsis, AtZAT18 and AtZAT10 enhance drought tolerance when overexpressed, though AtZAT10 overexpression also restricts plant growth [11,12]. AtZAT6 confers resistance to salt, drought, and bacterial pathogens [13,14]. In O. sativa, OsZFP213 and OsZFP179 improve salt tolerance [15,16], while OsZFP245 enhances drought tolerance by regulating stress-responsive genes [17]. In G. max, GmZAT4 participates in response to drought, salt, and abscisic acid (ABA) signaling [18]. These examples highlight the diverse functions of C2H2-ZFPs in plants.
Foxtail millet, an annual herbaceous crop belonging to the grass family (Poaceae), is an important grain crop in China, especially in northern regions [19,20]. Its seeds are rich in nutrients like protein, unsaturated fatty acids, dietary fiber, B vitamins, and essential minerals [21]. However, increasing abiotic stresses such as drought and salt have severely affected its growth and yield [22,23]. The seedling stage is particularly sensitive to these stresses, significantly influencing the crop’s overall development and productivity [24].
Despite the extensive research on C2H2-ZFPs in many plant species, no study has explored the C2H2-ZFP gene family in foxtail millet. In this study, we identified 67 C2H2-ZFP gene family members in the foxtail millet genome using bioinformatics methods. We analyzed their physical–chemical properties, chromosomal distribution, gene duplication events, and phylogenetic relationships. Moreover, we investigated the expression patterns of SiC2H2 genes under NaCl and drought stresses. This study provides the first systematic understanding of the C2H2-ZFP gene family in foxtail millet, laying a foundation for exploring stress tolerance regulatory mechanisms in foxtail millet.

2. Materials and Methods

2.1. Plant Materials and Treatments

The foxtail millet materials used in this study, Yugu 56, were provided by the Jilin Academy of Agricultural Sciences. Seeds with full grains were surface-sterilized sequentially with 75% alcohol (45 s) and 2% sodium hypochlorite solution (20 min), followed by rinsing with sterile water. The sterilized seeds were germinated on moist gauze for 48 h and then sown in pre-wetted nursery pots (substrate: vermiculite = 1:1), with 16 plants per pot. Seedlings were grown in a growth chamber under controlled conditions: temperature of 25 ± 2 °C, relative humidity of 70%, a photoperiod of 16 h light/8 h dark, and a light intensity of 2000 µmol·m−2·s−1. At the five-leaf stage (approximately 25 d), uniformly healthy seedlings were selected for abiotic stress treatment. Prior to treatment, seedling roots were rinsed with sterile water. The treatments included the following: (i) drought stress: 20% PEG-6000 solution; (ii) salt stress: 150 mmol/L NaCl solution. A sterile water treatment served as the control. For each treatment, three biological replicates were established, with five seedlings per replicate. Leaf samples were collected at 0 h and 24 h after treatment initiation, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.

2.2. Identification of C2H2 Genes in Foxtail Millet

The reference genome sequence for foxtail millet (Setaria italica v2.0) was obtained from the Ensembl Plants database (https://plants.ensembl.org/, accessed on 7 March 2025), which provides comprehensive genomic resources, including DNA, RNA, cDNA, and protein sequences. The protein sequence of the Arabidopsis C2H2 ZFPs was retrieved from the TAIR database (https://www.arabidopsis.org/, accessed on 7 March 2025). The C2H2 structural domain (PF00096) was acquired from the Pfam database (https://www.ebi.ac.uk/interpro/, accessed on 7 March 2025). To systematically identify C2H2-ZFP gene family members in foxtail millet, we performed a homology comparison of C2H2 protein sequences from Arabidopsis. By integrating the prediction results from two independent methods and removing redundant sequences, we identified 67 C2H2-ZFP gene family members. Subsequently, we conducted a comprehensive analysis of the physicochemical properties of the encoded proteins, including coding sequence length, amino acid composition, molecular weight (MW), theoretical isoelectric point (pI), and grand average of hydrophilicity (GRAVY), using the ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 7 March 2025). Additionally, subcellular localization predictions were performed using the Cell-PLoc 2.0 online platform (http://www.csbio.sjtu.edu.cn/, accessed on 7 March 2025).

2.3. Phylogenetic Tree, Chromosomal Location, and Conserved Motifs of SiC2H2

The full-length amino acid sequences of C2H2 genes from foxtail millet and Arabidopsis were aligned using the ClustalW algorithm implemented in MEGA11 software [25], with default alignment parameters. Based on the alignment results, a phylogenetic tree was constructed using the maximum likelihood (ML) method, with bootstrap analysis performed using 1000 replicates to assess node support [26]. All other parameters were maintained at default settings. The resulting phylogenetic tree was visualized and annotated using the Interactive Tree of Life (iTOL) online platform (https://itol.embl.de/, accessed on 10 March 2025) [27]. The chromosomal locations of the SiC2H2 genes were extracted from the foxtail millet genome FASTA and GFF3 files, with each gene systematically numbered based on its physical location on the chromosome. Conserved motifs within the SiC2H2 gene were identified using the MEME suite (https://meme-suite.org/meme/tools/meme, accessed on 10 March 2025), with the maximum number of motifs set to 10 and all other parameters maintained at default values [28]. Gene structure and conserved motifs were subsequently visualized and analyzed using TBtools software (v2.225) [29].

2.4. Whole-Genome Duplication and Synteny Analysis

Genome sequences and GFF3 files of Arabidopsis, rice, sorghum, and wheat were retrieved from the Ensembl Plants database (https://plants.ensembl.org/index.html, accessed on 7 March 2025). Genome-wide analyses were conducted using MCScanX to investigate genome-wide duplication (WGD) events, encompassing the identification of tandem duplicate genes, collinear blocks, and gene pairs [30]. For the identification of tandem duplicate genes, the following criteria were applied: (1) genes must reside on the same chromosome, with intergenic distances not exceeding 1000 bp; (2) sequence similarity between genes must exceed 85%; (3) the alignment length must cover more than 85% of the longest sequence [31,32]. Fragment duplications were detected using the BLASTN tool, with an E-value threshold set at 1 × 10−5 and a search window spanning 50 kilobases upstream and downstream of the coding sequence (CDS).

2.5. MiRNA and Cis-Acting Element Prediction

Using coding sequence (CDS) data of foxail millet C2H2 genes and published miRNA datasets [33], we employed the psRNATarget algorithm (https://www.zhaolab.org/psRNATarget, accessed on 10 March 2025) to identify putative miRNA binding sites [34]. This analysis predicted regulatory interactions between miRNAs and their target C2H2 genes. The promoter regions, spanning 2000 bp upstream of the start codon of each SiC2H2 gene, were extracted using TBtools software (v2.225). These promoter sequences were subsequently analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 March 2025) to identify cis-acting regulatory elements [35]. The systematic analysis of these elements aimed at environmental stresses. Finally, the distribution and types of cis-acting elements were visualized using TBtools.

2.6. Protein–Protein Interaction Network and GO Enrichment

The protein–protein interaction (PPI) network of C2H2-ZFP gene family members in foxtail millet was constructed using the STRING database (https://string-db.org/, accessed on 12 March 2025), with the parameters set to default values. The resulting PPI network was visualized using the R package (v4.4.3). Concurrently, gene ontology (GO) functional enrichment analysis of the SiC2H2 gene was performed using TBtools software (v2.225). The GO annotation was conducted by integrating the go-base.ob file from TBtools with the annotation results generated by the eggNOG-mapper online platform (http://eggnog-mapper.embl.de/, accessed on 12 March 2025). Finally, the GO enrichment results were visualized using TBtools, revealing the biological functions of the SiC2H2 gene and their involvement in the regulatory network.

2.7. Expression Pattern Analysis of SiC2H2

We integrated three RNA sequencing datasets of foxtail millet, including the following: (1) Yugu1 and An04 seedlings subjected to drought stress (EMBL-EBI Project No. PRJEB21225) [36] and (2) leaves of the salt-tolerant cultivar Hong Gu 2000 and salt-sensitive cultivar Pu Huang Yu under salt stress (NCBI Project No. PRJNA805389) [37]. Raw sequencing data were filtered and quality-controlled using fastp software (v0.25.0), followed by alignment to the foxtail millet reference genome (S. italica v2.0) using HISAT2. Gene expression quantification was performed using FeatureCounts (v2.0.6). Based on the expression data, heatmaps were generated using the heatmap package in R, enabling visualization of the expression patterns of the C2H2-ZFP gene family under various stress conditions and developmental stages in foxtail millet.

2.8. RNA Extraction, Reverse Transcription, and qRT-PCR Analysis

Total RNA was extracted from foxtail millet leaves using the Plant RNA Extraction Kit (Huayueyang Biotechnology Co., Ltd., Model GX, Beijing, China). RNA integrity and concentration were assessed by 1.5% agarose gel electrophoresis and a microspectrophotometer (Nanodrop ND-100, Premier Biosoft, Palo Alto, CA, USA). Reverse transcription of RNA to cDNA was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China), following the manufacturer’s instructions. qRT-PCR primers were designed using Primer Premier 6 software (Table S3), and quantitative fluorescence analysis was conducted using the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The reaction mixture (20 μL) consisted of 10 μL 2 × SYBR qPCR Master Mix, 2 μL cDNA template, 0.5 μL each of forward and reverse primers (10 μM), and 7 μL ddH2O. The SiActin gene was used as an internal reference to normalize target gene expression. Three biological replicates were included for each sample. The PCR program included the following steps: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 10 s; annealing at 54 °C for 30 s; extension at 65 °C for 10 s; and a final hold at 8 °C. Gene expression levels were calculated using the 2−ΔΔCT method [38].

2.9. Data Processing and Statistical Analysis

Data processing and statistical analysis were performed using Microsoft Excel (Microsoft, Redmond, WA, USA) and SPSS software (IBM, Armonk, NY, USA), respectively, while graphical representation was generated using Origin software (OriginLab, Northampton, MA, USA). The variability and uncertainty within the dataset were assessed using the standard deviation (STDEV) function.

3. Results

3.1. Genomic Identification, Chromosomal Localization, and Physicochemical Characterization of C2H2-ZFP Gene Family Members in Foxtail Millet

Through a dual screening approach combining HMM and Blast analyses, we identified 67 SiC2H2-ZFP genes in the foxtail millet genome. These genes were designated SiC2H2-1 to SiC2H2-67 based on their physical chromosomal positions. Chromosome distribution analysis revealed an uneven allocation across nine chromosomes, with chromosome IX harboring the highest gene density (16 genes) and chromosome IV the lowest (2 genes) (Figure 1). Analysis of physicochemical properties showed considerable variation in protein length, ranging from 865 amino acids (SiC2H2-1, the longest) to 89 amino acids (SiC2H2-49, the shortest). Molecular weights (MW) spanned 9.64~97.56 kDa, while isoelectric point (pI) analysis indicated that 70.15% (47/67) of the proteins were basic (pI > 7), suggesting enrichment in basic amino acid residues. The remaining 20 proteins (29.85%) were acidic (pI < 7), indicative of higher acidic residue content. Protein stability assessment revealed that most C2H2-ZFPs exhibited instability coefficients below 40 (range: 36.05~92.22), except for SiC2H2-50 and SiC2H2-51, implying reduced stability in vitro. The aliphatic index ranged from 46.36 to 78.66, while grand average of hydropathicity (GRAVY) index analysis classified 19 proteins as hydrophobic (GRAVY > −0.4). Subcellular localization predictions suggested nuclear localization for 65 members, with only SiC2H2-11 and SiC2H2-66 likely targeting chloroplasts (Table 1).

3.2. Phylogenetic and Evolutionary Analysis of C2H2-ZFPs Gene Family Members

To elucidate the evolutionary history of plant C2H2-ZFP genes, we analyzed 14 representative angiosperms (including foxtail millet). The C2H2 genes were widely distributed and abundant across plant species (Figure 2A), indicating strong evolutionary conservation in plants. We constructed a maximum likelihood phylogenetic tree of SiC2H2 genes in foxtail millet using Arabidopsis and rice as outgroups. The SiC2H2-ZFP genes clustered into seven branches (Clade I-IX), with notable absences in Clade I and V suggesting gene loss or functional divergence during foxtail millet evolution. Clade distribution analysis revealed the following: Clade II (3 genes, 4.48%); Clade III (12 genes, 17.91%); Clade IV and VII (17 genes each, 25.37%); Clade VI (4 genes, 5.97%); and Clade VIII and IX (7 genes each, 10.45%). The gene-rich Clades IV and VII may reflect genome duplication events. Comparative analysis showed that foxtail millet lacks Clade I and V members present in Arabidopsis and rice, implying functional remodeling during the adaptive evolution (Table S1 and Figure 2B).

3.3. SiC2H2 Gene Structure and Conserved Domains

Phylogenetic analysis of C2H2-ZFP genes in foxtail millet revealed six distinct clades, consistent with Figure 2B (Figure 3A). To elucidate functional domains, we predicted conserved motifs using MEME and visualized results using TBtools. The canonical C2H2 gene motif (Motif 1) was present in 97% (65/67) of members, absent only in SiC2H2-11 and SiC2H2-66. Motif numbers varied from 1 to 8 per protein: 4.5% (3/67) had 1 motif; 17.9% (12/67) had 2 motifs; 13.4% (9/67) had 3 motifs; 10.4% (7/67) had 4 motifs; and 53.7% (36/67) contained ≥ 5 motifs. Notably, genes with similar motif compositions clustered phylogenetically, indicating evolutionary conservation of domain architecture (Figure 3B). Exon–intron analysis further supported this trend, with exon numbers (1~8) correlating with clade membership. These results underscore structure–function coevolution in C2H2-ZFPs (Figure 3C).

3.4. Gene Duplication Events of the SiC2H2 Gene

Gene duplication analysis using Blast and MCScanX identified no tandem duplication in the foxtail millet C2H2-ZFP gene family. Circos plot visualization revealed 14 segmentally duplicated SiC2H2 gene pairs, with complete absence on chromosomes IV and VIII. Notably, SiC2H2-23 and SiC2H2-53 displayed exceptional duplication frequencies, forming collinear relationships with multiple loci (SiC2H2-56/57 and SiC2H2-32/65, respectively), while other members maintained single duplication pairs (Figure 4A).
Comparative genomic analysis demonstrated differential collinearity between foxtail millet C2H2 genes and related species: 22 gene pairs with Arabidopsis, 74 with rice, 76 with sorghum, and 163 with wheat. Whole-genome alignment showed that the foxtail millet C2H2 gene maintained collinearity with all chromosomes of Arabidopsis, rice, and wheat, except for chromosome 8 of sorghum. The limited collinearity with Arabidopsis (22 pairs) confirms their phylogenetic divergence, while the extensive conservation with wheat (163 pairs) suggests recent shared ancestry with Poaceae (Figure 4B).

3.5. Analysis of Cis-Acting Elements in Promoter of Foxtail Millet C2H2-ZFP Gene Family and miRNA Prediction

Analysis of the 2 kb promoter regions upstream of 67 foxtail millet SiC2H2 genes identified 1984 functional cis-acting elements after excluding core promoter elements (e.g., CAAT-box, and TATA-box) and low-frequency elements. These elements were classified into four functional categories: light-responsive (798 elements, 40.22%), hormone-responsive (781 elements, 39.36%), stress-responsive (303 elements, 15.27%), and developmental-related (102 elements, 5.14%). Among hormone-responsive elements, MeJA and abscisic acid-responsive elements were predominant (44.56% and 32.91%, respectively), while stress-responsive elements included anaerobic induction (28.38%), drought inducibility (23.76%), and low-temperature response (13.20%). Development elements were primarily associated with meristem expression (64.71%) and seed-specific regulation elements (35.29%). Notably, SiC2H2-3 and SiC2H2-5 contained the highest number of elements (55), whereas SiC2H2-1 had the lowest (10) (Table S2, Figure 5).
Integration of published foxtail millet miRNA data revealed that 10 SiC2H2 genes are potential miRNA targets, with SiC2H2-29 being the most extensively regulated (targeted by seven miRNAs). A ‘one-to-many’ regulation pattern was observed, exemplified by SitmiR156c targeting SiC2H2-5, SiC2H2-13, and SiC2H2-58 and SitmiR118d regulating both SiC2H2-44 and SiC2H2-45, suggesting miRNAs may coordinately fine-tune gene family modules (Table 2).

3.6. SiC2H2 Protein–Protein Interaction Network and GO Enrichment Analysis

Protein–protein interaction analysis of the C2H2-ZFP gene family in foxtail millet using the STRING database revealed seven significant interaction pairs. These included an interaction module comprising SiC2H2-8, SiC2H2-43, SiC2H2-20, and SiC2H2-6, along with four distinct pairwise interactions (SiC2H2-53/SiC2H2-10, SiC2H2-55/SiC2H2-17, SiC2H2-46/SiC2H2-25, and SiC2H2-21/SiC2H2-3). Notably, the interaction between SiC2H2-3 and SiC2H2-21 showed the highest confidence score, suggesting SiC2H2-21 may serve as a hub gene in the regulatory network (Figure 6A).
Functional characterization through GO enrichment analysis indicated these SiC2H2 proteins of foxtail millet primarily participate in fundamental biological processes, including regulation of primary metabolism process, regulation of nucleobase-containing compound metabolism process, regulation of gene expression, and DNA-templated transcription (Figure 6B).

3.7. SiC2H2 Gene Expression Pattern Analysis Based on RNA-Seq Data

Transcriptome analysis of two foxtail millet varieties (Hong Gu 2000 and Pu Huang Yu) revealed distinct expression patterns of SiC2H2 genes under salt stress (Figure 7A). While five genes (SiC2H2-5, 10, 29, 42, and 47) showed no expression across treatments, the salt-tolerant Hong Gu 2000 exhibited upregulation of 22 SiC2H2 genes, particularly SiC2H2-12, 35, 36, 58, and 63, contrasting with downregulation of SiC2H2-4, 51, 56, and 65. Pu Huang Yu displayed stress-induced upregulation of 25 genes, including SiC2H2-19, 20, 26, 31, 52, 60, 61, and 62, while 17 constitutively expressed genes were repressed post-stress.
Under drought conditions, four genes (SiC2H2-10, 29, 35, and 42) remained silent. Divergent expression patterns emerged between varieties, with SiC2H2-24/27 and SiC2H2-2/14 showing inverse regulation in Yugu vs. An04. Although ~70% of SiC2H2 genes were downregulated, specific members (SiC2H2-5, 11, 40, 41, and 48 in An04; SiC2H2-8, 15, 23, 32, 54, and 65 in Yugu) showed marked upregulation. These differential expression profiles suggest specialized functional diversification with the SiC2H2-ZFP family during abiotic stress responses (Figure 7B).

3.8. Expression Levels of SiC2H2 Gene in Foxtail Millet Under Abiotic Stress

While previous studies using NCBI datasets established the role of SiC2H2 genes in stress responses, we characterized cultivar-specific expression patterns in foxtail millet Yugu 56 through qPCR analysis. Under salt stress, eight SiC2H2 genes (SiC2H2-20/22/23/31/32/50/51/54) showed significant upregulation (P < 0.05), with SiC2H2-23 exhibiting the most pronounced induction (5.19-fold). In contrast, SiC2H2-30 was significantly repressed, while SiC2H2-53 showed non-significant upregulation.
Drought stress triggered similar upregulation patterns, except for SiC2H2-50 and SiC2H2-51, which were downregulated by 69.35% and 71.87%, respectively. The maximum induction under drought reached 5.47-fold. These findings both validate public datasets and reveal cultivar-specific regulatory mechanisms, providing a framework for functional studies of SiC2H2-mediated stress adaptation in millet (Figure 8).

4. Discussion

C2H2-ZFPs represent one of the largest transcription factor families in higher plants, playing crucial roles in growth, development, and stress response across diverse species, including Arabidopsis, rice, and soybean [39,40,41]. However, a comprehensive understanding of this gene family in foxtail millet has been lacking. Our genome-wide analysis identified 67 C2H2-ZFP genes in the S. italica v2.0 genome. Comparative analysis among 14 angiosperms showed that C2H2-ZFP family sizes in foxtail millet (67 genes), hemp (62), and grapes (64) were relatively conserved, contrasting sharply with the significantly expanded families in tobacco (247), soybean (321), and wheat (224) [42,43,44,45]. This variation is closely related to their polyploidization history. Foxtail millet, hemp, and grapes, lacking recent whole-genome duplication events [46,47], maintain smaller family sizes. In contrast, allopolyploid tobacco, which originated from ancestral hybridization and genome doubling, hexaploid wheat with its AABBDD genome, and soybean, which experienced two paleopolyploidization events around 59 and 13 million years ago, demonstrate how polyploidy drives the expansion of the C2H2-ZFP family [48]. This finding aligns with previous studies indicating that polyploidization often leads to the proliferation of transcription factor families, providing raw materials for subsequent functional divergence and adaptation [49,50,51].
Phylogenetic analysis of C2H2-ZFPs using the protein sequences from Arabidopsis, rice, and foxtail millet classified the foxtail millet C2H2-ZFP genes into seven subfamilies. Two subfamilies contained only members from Arabidopsis and rice, highlighting striking interspecific variation in subfamily organization. For example, C2H2-ZFPs in sorghum and Panax ginseng clustered into five subfamilies, while those in potato formed twelve subfamilies [9,52,53]. This taxonomic discrepancy can be attributed to the lack of unified classification criteria across studies and lineage-specific gene duplication and functional diversification during adaptation to different ecological niches. Although subfamily architectures vary among species, certain core subfamilies remain highly conserved, suggesting the preservation of essential biological functions. This conservation indicates that some fundamental regulatory pathways mediated by C2H2-ZFPs are evolutionarily stable. Meanwhile, the expansion of species-specific subfamilies and the variation in member counts within subfamilies reflect an evolutionary balance between maintaining conserved functions and evolving novel adaptations [5,54].
Genome-wide analysis revealed that the foxtail millet C2H2-ZFP gene family expanded mainly through segmental duplications, with 14 identified duplication events. This contrasts with the predominant tandem duplication patterns observed in sorghum (19 pairs) [9], potato (19 pairs) [53], B. oleracea (16 pairs) [55], and O. taihangensis (7 pairs) [56]. The different duplication mechanisms may be related to species-specific evolutionary histories and selective pressures. Comparative synteny analysis showed 163 colinear gene pairs between foxtail millet and wheat, significantly more than those between foxtail millet and rice (74 pairs), sorghum (76 pairs), or Arabidopsis (22 pairs). This pronounced genomic conservation strongly supports the hypothesis of shared polyploidization events in foxtail millet and wheat [57], which may have facilitated the retention of homologous genes and the conservation of gene regulatory networks related to important biological processes. Compared with other related studies, the high synteny between foxtail millet and wheat provides new insights into the evolution of the Poaceae family.
Analysis of promoter regions of 67 SiC2H2 genes identified 1967 cis-acting elements, predominantly related to light-responsive (40.21%), hormone-responsive (39.45%), and stress-responsive (29.45%) elements. This indicates that the SiC2H2 gene family is involved in photomorphogenesis, phytohormone signaling, and abiotic stress adaptation [58]. Notably, 10 SiC2H2 genes were predicted as miRNA targets, with the SiC2H2-29 gene regulated by seven distinct miRNAs, suggesting it has a potential role as a key regulatory node. Protein–protein interaction analysis identified seven significant SiC2H2 pairs, with SiC2H2-3/SiC2H2-21 showing the strongest interaction, implying the formation of a stable transcriptional complex to coordinate stress and hormone responses. GO enrichment analysis confirmed their roles in metabolic and transcriptional regulation. Integrating these results with previous studies, we can infer that the SiC2H2 transcription factors function within a complex regulatory network. miRNAs may fine-tune the expression of SiC2H2 genes in response to environmental changes, while protein complexes modulate their transcriptional activities, jointly shaping the plant’s growth, development, and stress responses.
RNA-seq data analysis of foxtail millet revealed distinct expression patterns of SiC2H2 genes under abiotic stress. In salt-tolerant varieties, SiC2H2-35 and SiC2H2-58 genes were significantly upregulated under salt stress, potentially coordinating shoot architecture maintenance and root function modulation to enhance whole-plant salt tolerance. In contrast, in salt-sensitive varieties, SiC2H2-19, SiC2H2-52, and SiC2H2-60 showed elevated expression, possibly representing ineffective compensatory responses due to disrupted regulatory networks. For drought stress, tolerant genotypes utilized specific SiC2H2 genes to regulate stomatal regulation, cuticular wax deposition, osmoprotectant synthesis, and growth, establishing an integrated drought defense system. In contrast, drought-sensitive varieties exhibited maladaptive overexpression of SiC2H2-11 (disrupting photomorphogenesis) and SiC2H2-41 (inducing excessive growth suppression), increasing their drought susceptibility. These results substantiate the pivotal role of C2H2-ZFPs in abiotic stress adaptation [59,60]. These findings are consistent with previous reports on the role of C2H2-ZFPs in abiotic stress responses in other plants. However, the specific genes and regulatory mechanisms may vary among species, reflecting the species-specific adaptation strategies. Our results further emphasize the importance of C2H2-ZFPs in abiotic stress adaptation and provide potential gene targets for breeding stress-tolerant foxtail millet varieties.

5. Conclusions

Our genome-wide analysis identified 67 C2H2-ZFP genes in foxtail millet, revealing their conserved roles in light, hormone, and stress signaling pathways. Functional characterization of the Yugu 56 cultivar demonstrated significant stress-induced upregulation of SiC2H2-23 (5.19-fold under salt stress) and Si2H2-32 (5.47-fold under drought), highlighting their potential as genetic markers for stress resilience. As the first comprehensive study of C2H2-ZFPs in foxtail millet, these findings establish a molecular framework for understanding abiotic stress responses in foxtail millet and provide targets for precision breeding approaches.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15071618/s1, Table S1: The amino acid sequences of SiC2H2 protein in foxtail millet; Table S2: The number and corresponding relationship of cis-acting elements of the SiC2H2 gene family in foxtail millet; Table S3: Primers of SiC2H2 gene for qPCR analysis.

Author Contributions

Conceptualization, Q.Z., Y.Z., X.X., Z.Z. and J.Z. (Jian Zhang); methodology, Q.Z., Y.Z., X.X., J.Z. (Jun Zhang), L.J., Z.Z., M.G. and J.Z. (Jian Zhang); software, Q.Z., Y.Z., X.X. and S.L.; validation, R.S., W.Z. and J.Z. (Jun Zhang); investigation, Q.Z., X.X. and L.J.; resources, W.Z. and M.G.; data curation, Q.Z., Y.Z., S.L. and R.S.; writing—original draft preparation, Q.Z.; writing—review and editing, Q.Z., Z.Z., M.G. and J.Z. (Jian Zhang); visualization, Q.Z. and M.G.; supervision, Z.Z. and J.Z. (Jian Zhang); project administration, Z.Z.; funding acquisition, Z.Z. and M.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jilin Provincial Science and Technology Development Program Project (No. 20240303010NC).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We would like to express our gratitude to the Jilin Academy of Agricultural Sciences for providing the Yugu 56 materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Chromosomal distribution of SiC2H2 genes in foxtail millet. Chromosomes are color-coded by gene density (blue: low; red: high), The Roman numerals I, II, etc. represent chromosome numbers.
Figure 1. Chromosomal distribution of SiC2H2 genes in foxtail millet. Chromosomes are color-coded by gene density (blue: low; red: high), The Roman numerals I, II, etc. represent chromosome numbers.
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Figure 2. Phylogenetic and evolutionary analysis of SiC2H2-ZFP genes in foxtail millet. (A) Comparative analysis of C2H2-ZFP gene counts across angiosperm species. (B) Maximum likelihood phylogenetic trees of C2H2-ZFP members in Arabidopsis, rice, and foxtail millet. Branches are color-coded by clade. The stars represent members of the SiC2H2 gene family in foxtail millet.
Figure 2. Phylogenetic and evolutionary analysis of SiC2H2-ZFP genes in foxtail millet. (A) Comparative analysis of C2H2-ZFP gene counts across angiosperm species. (B) Maximum likelihood phylogenetic trees of C2H2-ZFP members in Arabidopsis, rice, and foxtail millet. Branches are color-coded by clade. The stars represent members of the SiC2H2 gene family in foxtail millet.
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Figure 3. Phylogenetic tree, conserved motifs, and exon–intron structure analysis of SiC2H2 genes in foxtail millet. (A) Phylogenetic tree of SiC2H2 genes in foxtail millet. (B) Conserved motifs of SiC2H2 proteins. Ten motifs are color-coded for visualization. (C) Exon–intron structures of SiC2H2 genes in foxtail millet.
Figure 3. Phylogenetic tree, conserved motifs, and exon–intron structure analysis of SiC2H2 genes in foxtail millet. (A) Phylogenetic tree of SiC2H2 genes in foxtail millet. (B) Conserved motifs of SiC2H2 proteins. Ten motifs are color-coded for visualization. (C) Exon–intron structures of SiC2H2 genes in foxtail millet.
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Figure 4. Collinearity analysis of SiC2H2 genes. (A) Intra-genomic collinear gene pairs in foxtail millet (In the Circos plot, the connecting lines represent intra-genomic collinear gene pairs in foxtail millet). (B) Inter-species collinearity between foxtail millet and four representative species (Arabidopsis, rice, sorghum, and wheat). Gray lines denote background synteny, while red lines highlight collinear SiC2H2 gene pairs.
Figure 4. Collinearity analysis of SiC2H2 genes. (A) Intra-genomic collinear gene pairs in foxtail millet (In the Circos plot, the connecting lines represent intra-genomic collinear gene pairs in foxtail millet). (B) Inter-species collinearity between foxtail millet and four representative species (Arabidopsis, rice, sorghum, and wheat). Gray lines denote background synteny, while red lines highlight collinear SiC2H2 gene pairs.
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Figure 5. Cis-acting element analysis of foxtail millet SiC2H2 genes. (A) Genome-wide distribution of cis-regulatory elements in SiC2H2 promoters. (B) Classification and abundance of element types. (C) Quantitative analysis of element counts per promoter region.
Figure 5. Cis-acting element analysis of foxtail millet SiC2H2 genes. (A) Genome-wide distribution of cis-regulatory elements in SiC2H2 promoters. (B) Classification and abundance of element types. (C) Quantitative analysis of element counts per promoter region.
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Figure 6. Protein interaction network and functional enrichment analysis of C2H2-ZFPs in foxtail millet. (A) Predicted protein–protein interaction network of SiC2H2 members. (B) Gene ontology (GO) term enrichment profile for SiC2H2 genes.
Figure 6. Protein interaction network and functional enrichment analysis of C2H2-ZFPs in foxtail millet. (A) Predicted protein–protein interaction network of SiC2H2 members. (B) Gene ontology (GO) term enrichment profile for SiC2H2 genes.
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Figure 7. Expression profiles of SiC2H2 genes under abiotic stress conditions. (A) Salt stress responses in two foxtail millet cultivars: PHY−Salt (salt-treated ‘Pu Huang Yu’), PHY−CK (untreated control), HG−Salt (salt-treated ‘Hong Gu 2000’), and HG−CK (untreated control). (B) Drought stress responses in cultivars An04−Drought (stress-treated) vs. An04−CK (control) and YG−Drought (stress-treated ‘Yugu 1’) vs. YG−CK (control).
Figure 7. Expression profiles of SiC2H2 genes under abiotic stress conditions. (A) Salt stress responses in two foxtail millet cultivars: PHY−Salt (salt-treated ‘Pu Huang Yu’), PHY−CK (untreated control), HG−Salt (salt-treated ‘Hong Gu 2000’), and HG−CK (untreated control). (B) Drought stress responses in cultivars An04−Drought (stress-treated) vs. An04−CK (control) and YG−Drought (stress-treated ‘Yugu 1’) vs. YG−CK (control).
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Figure 8. Expression levels of the SiC2H2 gene under salt and drought stress in foxtail millet (Yugu 56). CK: control (non-stressed condition); S24: salt stress treatment (150 mmol/L NaCl) for 24 h; D24: drought stress treatment (20% PEG-6000) for 24 h. Yellow represents CK; Red represents salt stress treatment for 24 hours; Green represents drought stress treatment for 24 hours. Data were analyzed by two-way ANOVA in GraphPad Prism. Significance levels are indicated as follows: ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 8. Expression levels of the SiC2H2 gene under salt and drought stress in foxtail millet (Yugu 56). CK: control (non-stressed condition); S24: salt stress treatment (150 mmol/L NaCl) for 24 h; D24: drought stress treatment (20% PEG-6000) for 24 h. Yellow represents CK; Red represents salt stress treatment for 24 hours; Green represents drought stress treatment for 24 hours. Data were analyzed by two-way ANOVA in GraphPad Prism. Significance levels are indicated as follows: ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Table 1. Identification and characterization prediction of SiC2H2 gene in Foxtail millet.
Table 1. Identification and characterization prediction of SiC2H2 gene in Foxtail millet.
Gene NameLength (aa)MW (KDa)pIInstability IndexAliphatic IndexGrand Average of Hydropathicity (GRAVY)
SiC2H2-186597.567.6840.5978.66−0.298
SiC2H2-248853.597.6459.6663.01−0.633
SiC2H2-335039.447.9655.1753.6−0.957
SiC2H2-439241.626.2557.4364.44−0.442
SiC2H2-556960.545.9653.7168.49−0.417
SiC2H2-634035.136.0664.1354.56−0.44
SiC2H2-758560.218.8952.8759.2−0.4
SiC2H2-827128.918.2270.7853.47−0.577
SiC2H2-931532.778.6561.4170.51−0.3
SiC2H2-1030030.519.2761.5368.17−0.32
SiC2H2-1114016.0510.9769.5353.79−0.689
SiC2H2-1234837.666.6757.7852.21−0.565
SiC2H2-1347150.409.2171.3263.99−0.537
SiC2H2-1443346.666.0658.6253.37−0.749
SiC2H2-1551152.809.1749.4855.03−0.471
SiC2H2-1671178.536.9373.5652.74−0.86
SiC2H2-1718719.258.9373.9265.99−0.243
SiC2H2-1841943.705.8969.3360.02−0.563
SiC2H2-1937842.928.8455.7752.06−0.873
SiC2H2-2018820.126.7841.8372.77−0.232
SiC2H2-2137842.407.6551.2648.78−0.981
SiC2H2-2239042.817.2353.3157.36−0.67
SiC2H2-2325726.637.6492.2264.12−0.375
SiC2H2-2439342.207.2752.6246.36−0.78
SiC2H2-2518218.899.0569.1166.21−0.295
SiC2H2-2640242.799.3654.8671.02−0.388
SiC2H2-2746549.369.0448.7657.7−0.529
SiC2H2-2843646.368.9454.1555.53−0.559
SiC2H2-2949751.145.5460.2664.08−0.429
SiC2H2-3047450.158.7956.8657.45−0.556
SiC2H2-3121423.149.0469.4775.42−0.481
SiC2H2-3218419.376.1668.7858.1−0.622
SiC2H2-3351756.735.6246.2959.81−0.616
SiC2H2-3451756.735.6246.2959.81−0.616
SiC2H2-3545447.366.672.5858.33−0.595
SiC2H2-3646949.758.8653.860.43−0.464
SiC2H2-3741945.235.6657.2657.21−0.492
SiC2H2-3852954.709.4448.7153.69−0.53
SiC2H2-3952654.449.3349.8453.99−0.522
SiC2H2-4039241.918.754.6963.16−0.42
SiC2H2-4150351.995.8357.3263.12−0.477
SiC2H2-4256259.076.1854.3163.79−0.423
SiC2H2-4331933.317.0966.1755.92−0.571
SiC2H2-4460762.568.7745.5658.07−0.442
SiC2H2-4560762.568.7745.5658.07−0.442
SiC2H2-4647752.215.9945.559.92−0.534
SiC2H2-4730431.449.6275.8165.03−0.471
SiC2H2-4813414.717.0942.6767.16−0.754
SiC2H2-49899.6410.2766.3172.36−0.319
SiC2H2-5016517.5410.3637.0272.85−0.351
SiC2H2-5114214.7810.1636.0571.69−0.149
SiC2H2-5233136.416.3150.7564.92−0.463
SiC2H2-5323924.8710.0454.8371.21−0.313
SiC2H2-5418619.838.4359.2463.71−0.565
SiC2H2-5514214.677.8270.1769.08−0.389
SiC2H2-5640842.899.1867.9364.73−0.346
SiC2H2-5723024.479.1478.5261.78−0.489
SiC2H2-5839942.939.4852.171.33−0.396
SiC2H2-5934036.418.9881.147.74−0.791
SiC2H2-6022423.128.5666.5568.62−0.197
SiC2H2-6122023.058.763.9972.09−0.217
SiC2H2-6220821.399.369.0365.48−0.249
SiC2H2-6340543.955.7265.7960.35−0.757
SiC2H2-6414415.176.9653.2472.08−0.419
SiC2H2-6518819.897.0946.1470.69−0.401
SiC2H2-6645448.1410.9575.9967.44−0.502
SiC2H2-6753755.82971.5860.32−0.383
Note: SiC2H2-11 and SiC2H2-66 are localized in chloroplasts, while the remaining SiC2H2 proteins are localized in the nucleus.
Table 2. miRNA target prediction of SiC2H2 gene.
Table 2. miRNA target prediction of SiC2H2 gene.
MiRNATargetExpectationMiRNA LengthTarget_StartTarget_End
SitmiR156cSiC2H2-584.02294115
SitmiR156cSiC2H2-55.022458479
SitmiR156cSiC2H2-135.0229951016
SitmiR166a-1/2/3/4/5SiC2H2-294.02113131333
SitmiR166c/dSiC2H2-294.02113131333
SitmiR2118a-1/2/3SiC2H2-24.52212981319
SitmiR2118dSiC2H2-444.52212491270
SitmiR2118dSiC2H2-454.52212491270
SitmiR164a/bSiC2H2-505.021164184
SitmiR166a-1/2/3SiC2H2-185.0211131
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Zhao, Q.; Zhang, Y.; Xing, X.; Li, S.; Sun, R.; Zhang, W.; Zhang, J.; Jiang, L.; Zang, Z.; Gao, M.; et al. Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica). Agronomy 2025, 15, 1618. https://doi.org/10.3390/agronomy15071618

AMA Style

Zhao Q, Zhang Y, Xing X, Li S, Sun R, Zhang W, Zhang J, Jiang L, Zang Z, Gao M, et al. Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica). Agronomy. 2025; 15(7):1618. https://doi.org/10.3390/agronomy15071618

Chicago/Turabian Style

Zhao, Qian, Yingxin Zhang, Xiangyu Xing, Shuyao Li, Ruidong Sun, Weilong Zhang, Jun Zhang, Liangyu Jiang, Zhenyuan Zang, Ming Gao, and et al. 2025. "Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica)" Agronomy 15, no. 7: 1618. https://doi.org/10.3390/agronomy15071618

APA Style

Zhao, Q., Zhang, Y., Xing, X., Li, S., Sun, R., Zhang, W., Zhang, J., Jiang, L., Zang, Z., Gao, M., & Zhang, J. (2025). Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica). Agronomy, 15(7), 1618. https://doi.org/10.3390/agronomy15071618

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