Overexpression of a White Clover WRKY Transcription Factor Improves Cold Tolerance in Arabidopsis

Abstract

Plants are frequently exposed to various abiotic stresses, among which low-temperature stress markedly impairs growth and physiological functions. WRKY transcription factors are key regulators in plant responses to abiotic stress. In this study, a novel WRKY transcription factor gene, TrWRKY79, was cloned from white clover. Functional characterization revealed that the full-length TrWRKY79 protein possesses typical features of transcription factors, including transcriptional activation activity located at its C-terminal domain. Heterologous expression of TrWRKY79 in Arabidopsis thaliana significantly enhanced cold tolerance under low-temperature stress. Physiological assays showed that the transgenic lines exhibited higher chlorophyll content and elevated activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) compared to wild-type plants. Furthermore, Protenix was employed to predict the potential target genes of TrWRKY transcription factors, and their expression profiles were analyzed to help elucidate the regulatory network underlying cold tolerance. qRT-PCR analysis confirmed that several cold-responsive genes, such as COR47 and ABI5, were significantly upregulated in the transgenic lines. Collectively, these findings indicate that TrWRKY79 plays a positive regulatory role in enhancing cold tolerance, providing valuable insights into the molecular mechanisms of cold resistance in white clover and offering promising candidate genes for improving stress resilience in forage crops.

1. Introduction

Low-temperature stress constitutes a major abiotic constraint during plant growth and development, severely disrupting physiological metabolism through membrane structural damage, enzymatic activity reduction, photosynthetic inhibition, and growth arrest, ultimately leading to plant mortality under extreme conditions [1]. These physiological impairments ultimately result in crop yield reduction and quality deterioration, posing significant threats to agricultural productivity and food security. Through evolutionary adaptation, plants have developed sophisticated molecular response mechanisms, encompassing cold signal perception, signaling pathway activation, transcriptional regulation of stress-responsive genes, and metabolic reprogramming, to mitigate chilling damage [2].

Within this regulatory framework, transcription factors (TFs) serve as pivotal nodal components of gene networks, exerting central regulatory roles through their recognition of specific cis-acting elements in target gene promoters to modulate the expression of cold-responsive genes [3]. These regulatory proteins not only control the spatial and temporal patterns of gene expression but also mediate the dynamic response of plants to environmental fluctuations and play a crucial role in abiotic stress adaptation. Several studies have shown that various TF families, including bHLH, bZIP, MYB, NAC, and WRKY, are coordinately involved in the coordination of cold stress signaling networks [4,5,6,7,8]. These TFs regulate critical physiological processes, such as antioxidant system activation, osmolyte biosynthesis, and membrane stabilization. For instance, Liu et al. identified OsbZIP52 as a regulatory component enhancing cold stress sensitivity through overexpression in rice, while Zhang’s team revealed that it improves cold tolerance in transgenic cucumber plants through ABA signaling-dependent mechanisms [9,10]. The WRKY family has emerged as a focal research domain in plant cold response mechanisms due to its evolutionarily conserved structure coupled with functional diversification.

WRKY transcription factors constitute a plant-specific regulatory protein family characterized by functional versatility, ubiquitously distributed in higher plants as one of the largest and most functionally complex TF families [11]. Initially identified in sweet potato as SPF1 (Sweet Potato Factor 1), WRKY genes have since been extensively studied and classified in model organisms, including Arabidopsis and rice [12,13,14]. The structural hallmark of WRKY proteins involves one or two WRKY domains—each containing the conserved WRKYGQK motif paired with C2H2 or C2HC-type zinc finger motifs—which form the molecular basis for their biological functions [15]. Based on domain architecture and zinc finger configuration, WRKY proteins are categorized into three major groups: Group I contains dual WRKY domains, while Groups II and III possess single domains distinguished by their zinc finger structural variations [16]. The WRKY domain specifically recognizes W-box cis-elements (core sequence TTGACC/T) in gene promoters, enabling transcriptional regulation of diverse physiological processes. Mechanistically, WRKY TFs enhance cold tolerance by modulating target genes involved in osmolyte biosynthesis, antioxidant system activation, and membrane composition remodeling through W-box recognition [17]. Numerous studies document significant cold-induced upregulation of WRKY genes and their regulatory effects on downstream cold-responsive genes. In Arabidopsis, WRKY34 and WRKY33 participate in pollen development and ROS scavenging, respectively, both functionally linked to cold stress responses [18,19]. Rice OsWRKY76 demonstrates cold-inducible expression that promotes antioxidant enzyme production and lipid transfer protein synthesis, thereby reinforcing membrane stability and chilling tolerance [20]. Notably, transgenic Arabidopsis overexpressing wheat TaWRKY19 exhibits enhanced cold resistance through upregulated expression of stress-responsive genes, including DREB2A, RD29A, RD29B, and COR6.6 [21]. These findings collectively underscore WRKY TFs’ pivotal roles in orchestrating molecular networks underlying plant cold adaptation mechanisms. Collectively, WRKY transcription factors exhibit multilayered regulatory capacity across multiple pathways, functioning as indispensable molecular switches in plant cold signaling networks.

Although WRKY proteins have been demonstrated to participate in low-temperature stress regulation across numerous plant species, their regulatory mechanisms in white clover remain systematically uncharacterized. White clover, a perennial legume widely distributed in temperate and cold-temperate regions, is extensively cultivated for its high productivity, superior quality, and robust environmental adaptability. It is broadly utilized in landscape greening, forage production, and medicinal applications because of its pharmacological properties, such as heat-clearing and phlegm-removing functions [22]. However, its growth is severely inhibited under conditions of abiotic stress, such as high salinity, drought, and low temperatures, especially at high latitudes where winters are severe, leading to widespread mortality and significant limitations in cultivation and agricultural production. Consequently, enhancing cold tolerance in white clover has become a critical challenge for improving its cultivation efficiency. To elucidate its cold adaptation mechanisms, this study cloned the TrWRKY79 gene from white clover and introduced it into the model plant Arabidopsis thaliana for systematic functional analysis under low-temperature stress. This research not only provides novel insights into the molecular basis of cold resistance in white clover but also identifies a potential high-quality genetic resource for molecular breeding programs, offering substantial theoretical significance and practical prospects for agricultural innovation.

2. Materials and Methods

2.1. Plant Materials and Stress Treatments

This study employed white clover (cultivar Haifa, sourced from Barenbrug China Ltd. Com.) (Beijing, China) as the principal biological substrate. All sprouted seeds were transferred to a substrate mixture containing perlite and sand in a 3:1 volumetric ratio [23]. Seedlings were maintained in individual pots with a density of 10–15 plants per cultivation unit. The cultivation parameters included 24 °C/18 °C day/night temperature regimes and alternate-day supplementation with 50% Hoagland nutrient solution. Following a 28-day growth period, cold stress exposure protocols were implemented. Leaf samples were harvested after 24 h of 4 °C treatment under controlled environmental parameters. The collected samples underwent immediate liquid nitrogen immersion prior to long-term storage at −80 °C. In addition, Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild-type (WT) control in all experiments. Arabidopsis seeds were cultivated in a greenhouse under controlled environmental conditions: a temperature of 24 °C, a photoperiod of 16 h of light/8 h of dark, and a relative humidity of about 60%. The cultivation utilized a standardized substrate containing equal volumes of peat moss and vermiculite.

2.2. Cloning of TrWRKY79 Gene and Sequence Analysis

Total RNA was extracted from cold-stressed (4 °C) white clover leaves using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). RNA concentration quantification was conducted using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA quality and integrity were assessed through 1% agarose gel electrophoresis. Reverse transcription into cDNA templates was performed using the PrimeScript RT Kit (Toyobo, Shanghai, China). Full-length TrWRKY79 cDNA was amplified by PCR with the gene-specific primers detailed in

Table A1. Primers for TrWRKY79 cloning and vector linkage.

Primer Name	Primer Sequence (5′-3′)
TrWRKY79-F	AATCAACCAACCCTATAT
TrWRKY79-R	ATGAATATGATTGTGTTAT
BD-TrWRKY79-F	CATGGAGGCCGAATTCAATCAACCAACCCTATAT
BD-TrWRKY79-R	GGATCCCCGGGAATTCATGAATATGATTGTGTTAT
BD-TrWRKY79-N-F	CATGGAGGCCGAATTCAATCAACCAACCCTATAT
BD-TrWRKY79-N-R	GGATCCCCGGGAATTCTCCAGATGATCCACCTCGCT
BD-TrWRKY79-W-F	CATGGAGGCCGAATTCAGCGAGGTGGATCATCTGGA
BD-TrWRKY79BW-R	GGATCCCCGGGAATTCAGCCATAACTGGATTTGGATGAGTA
BD-TrWRKY79-C-F	CATGGAGGCCGAATTCTACGAAGGGAAACATACTC
BD-TrWRKY79-C-R	GGATCCCCGGGAATTCATGAATATGATTGTGTTAT

. Purified amplification products were cloned into pMD18-T vectors (TaKaRa) for sequencing analysis. The Arabidopsis WRKY protein sequence was downloaded using TAIR (https://www.arabidopsis.org) (accessed on 3 January 2024). The phylogenetic tree of TrWRKY79 and Arabidopsis WRKY genes was constructed using MEGA11, the neighbor-joining method, and 1000 bootstrap repeats [24].

2.3. Transactivation Assay of TrWRKY79

Domain architecture prediction via SMART database partitioned TrWRKY79 into three segments: TrWRKY79-N (1–139 aa), TrWRKY79-W (Structural domain, 140–198 aa), and TrWRKY79-C (199–292 aa). Full-length TrWRKY79 and its three truncated fragments were cloned into a pGBKT7 (BD) vector via a homologous recombination strategy. The primer sequences are detailed in Appendix A, Table A1. The recombinant plasmids (BD-TrWRKY79, -N, -W, -C) were transformed into yeast strain AH109, with an empty pGBKT7 vector serving as the negative control. Both the recombinant plasmids and negative controls were separately transformed into yeast competent cells. The transformed yeast colonies were streaked on SD/-Trp and SD/-Trp/-His plates, followed by 3–5 days incubation at 30 °C for growth observation.

2.4. Arabidopsis Transformation and Transgenic Plant Validation

The recombinant vector pCAMBIA1300-TrWRKY79 was constructed through BamHI/PstI double digestion of pMD18T-TrWRKY79 and pCAMBIA1300 plasmids, followed by T4 DNA ligase-mediated insertion of purified target fragments. The constructed recombinant vector was introduced into Agrobacterium tumefaciens GV3101 via the freeze–thaw transformation method. Thirty-day-old robust Arabidopsis plants were selected for floral dip transformation after removing opened inflorescences and siliques, followed by cultivation to seed maturity for T0 generation collection [25].

Mature T0 seeds with full maturity were surface-sterilized by sequential treatment: immersion in 75% ethanol for 30 s, followed by 1% sodium hypochlorite for 5 min. After sterilization, the seeds were rinsed 5–6 times with sterile distilled water to remove residual disinfectants. The sterilized seeds were stratified on ½ MS medium containing kanamycin (50 mg/L) at 4 °C for 2 days to break dormancy and then transferred to a growth chamber under standard conditions (24 °C, 60% RH, 16/8 h photoperiod). Kanamycin selection was continued for an additional 2 days to effectively eliminate non-transformed seedlings while allowing initial establishment of transformed individuals. Two-week-old seedlings exhibiting normal growth phenotypes were transplanted into nutrient soil-containing pots. PCR validation using Arabidopsis leaf DNA templates was conducted, with amplicons matching the expected sizes submitted to BGI Genomics for sequencing verification. Positive transgenic lines were advanced through successive generations (T1–T3) using the described protocol to obtain homozygous T3 plants for experimentation. Both wild-type and T3 transgenic plants were subjected to 24h cold stress (4 °C, 16/8 h photoperiod) after a 4-week identical cultivation, with phenotypic alterations documented systematically.

A 24 h cold treatment was selected to induce early-stage transcriptional and physiological responses. WRKY transcription factors are known to function as early responders to abiotic stresses, such as cold, and can quickly activate downstream stress-responsive genes. Our preliminary qRT-PCR results indicated that the expression of cold-responsive marker genes, such as AtCOR47 and AtERD10, peaked around 24 h after cold exposure, suggesting this duration effectively captures WRKY-mediated regulatory effects. Similar short-term cold treatments have been employed in previous studies. For instance, Li et al. used a 12 h 4 °C treatment to examine the role of VhWRKY44 in transgenic Arabidopsis [26].

2.5. Physiological and Biochemical Characterization of TrWRKY79-Transgenic Arabidopsis

Following 4 weeks of cultivation per Method 2.4, wild-type (WT) and T3 transgenic Arabidopsis plants were divided into two experimental groups: a control group, maintained under normal conditions, and a treatment group, exposed to 24 h cold stress at 4 °C with a 16/8 h light/dark cycle. Tissue samples were collected, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent quantification of chlorophyll (CHL), proline (Pro), malondialdehyde (MDA), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) levels, and expression analysis of stress-responsive genes. The chlorophyll concentration was determined by the acetone extraction method [27]. The content of malondialdehyde (MDA) was determined using the thiobarbituric acid (TBA) assay. Proline levels were evaluated through a ninhydrin-based colorimetric method. Enzymatic activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were analyzed spectrophotometrically according to the procedure outlined by Wang et al. [28]. All physiological assays were performed with three biological replicates, and data are presented as mean ± standard deviation (SD).

2.6. TrWRKY79 Transcription Factor Binding Site

To investigate the regulatory mechanisms of WRKY transcription factors, W-box elements were identified and extracted from the promoter regions of Arabidopsis thaliana. A genome-wide scan was performed using the conserved core W-box motif TTGAC[C/T] as the search pattern. Promoter sequences comprising 2000 base pairs upstream of all annotated genes were retrieved from the TAIR database (https://www.arabidopsis.org). A custom Python script (version 3.9) employing a regular expression matching algorithm was used to detect potential WRKY binding sites. For each identified W-box, 5 nucleotides upstream and downstream were extracted to obtain extended sequences ranging from 10 to 15 base pairs. These extended sequences were then analyzed for frequency distribution, and the 20 most frequently occurring motifs were selected as representative W-box elements (Supplements2_CRE.py). The target sequences were converted into FASTA format and subjected to BLAST analysis (E-value threshold of 2) against the Arabidopsis promoter database to predict potential target genes under transgenic conditions. Considering the properties of the W-box core motif (TTGAC[C/T]) and its extended flanking sequences, this E-value threshold represents a balanced choice between alignment sensitivity and specificity, similar to microRNA (21–24 nt) search works [29,30]. A total of 100 nucleotide sequences containing W-boxes were predicted based on BLAST results, selection of optimal e-values, and literature references. Potential interrelationships of these sequences with the WRKY79 protein were predicted using Protenix [31]. Selected protein–DNA interaction models were visualized using ChimeraX [32].

2.7. Expression Analysis of Resistance-Related Genes in Transgenic Arabidopsis thaliana

After four weeks of growth, total RNA was extracted from both the wild-type (WT) and transgenic homozygous Arabidopsis thaliana. The samples were divided into non-stressed and cold-stressed groups. RNA was reverse-transcribed into cDNA using the PrimeScript RT kit (Toyobo, Shanghai, China), which was used as a template for quantitative real-time PCR (qRT-PCR). Expression levels of selected downstream target genes regulated by WRKY transcription factors were quantified using a SYBR-based qRT-PCR system. Based on predictions from Protenix (Section 2.6), five candidate genes—AtCOR47, AtABI5, AtRAB18, AtCOR15A, and AtERD10—were selected for expression validation, using the gene-specific primers listed in

Table A2. Primer sequences for qRT-PCR analysis.

Primer Name	Primer Sequence (5′-3′)
qTrWRKY79-F	TATCCTGTGGATGATGCAGT
qTrWRKY79-R	GATCCACCTCGCTTTTAGTC
qAtactin-F	AGCTAGAGACAGCCAAGAGC
qAtactin-R	GCTTCCATTCCGATGAGCGA
qAtCOR47-F	GAAACCTCAAGAGACAACGA
qAtCOR47-R	AGAAGAGCTGTTGGATCGG
qAtABI5-F	TCCAGTGGAGAAAGTAGTGG
qAtABI5-R	AGGTGTCTTTACCTGTTGCT
qAtRAB18-F	GTCAGGACAACCCGAATTT
qAtRAB18-R	ATACGAACTTGTTAGCGTCC
qAtCOR15A-F	GAACAAGCCTAGTGTCATCG
qAtCOR15A-R	ATCATCCTCTGCTGTCTTGT
qAtERD10-F	ACATTCGGTAGAGGATCACA
qAtERD10-R	TTCCTCTCCAGTGGTCTTG

. AtActin was used as the internal reference gene for normalization of gene expression levels, and each experiment was performed with three biological replicates. qRT-PCR assays were performed using the LightCycler 96 system (Roche, Rotkreuz, Switzerland) and SYBR Premix Ex Taq™ II (Toyobo, Shanghai, China), with a reaction volume of 20 µL per sample. The qPCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Relative gene expression levels were calculated using the 2^−ΔΔCT method [33].

3. Results

3.1. Cloning and Sequence Analysis of TrWRKY79

The TrWRKY79 gene was amplified from white clover cDNA by PCR using gene-specific primers TrWRKY79-F1 and TrWRKY79-R1 (Table A1). The expected amplicon was confirmed by agarose gel electrophoresis, as shown in Appendix B, Figure A1. NCBI analysis revealed that the open reading frame (ORF) of TrWRKY79 is 877 base pairs in length, encoding a protein consisting of 292 amino acids. Based on ProParam predictions, the encoded protein has a molecular formula of C₁₄₀₂H₂₂₀₂N₃₉₈O₄₅₇A1₄, with a molecular weight of 32,394.14 kDa and a theoretical isoelectric point (pI) of 6.26. The instability index was calculated as 54.43, indicating that TrWRKY79 is an unstable and acidic protein. The protein consists of 20 amino acid types, with serine (12.10%), asparagine (8.00%), and leucine (8.00%) being the most abundant. It contains 31 positively charged residues (arginine and lysine) and 34 negatively charged residues (aspartic acid and glutamic acid). The aliphatic index is 63.08, the theoretical half-life is estimated to be 30 h, and the grand average of hydropathicity (GRAVY) is 0.746. Subcellular localization prediction using WoLF PSORT suggested that TrWRKY79 is primarily localized in the nucleus (78.7%), with minor distributions in the chloroplast, plasma membrane, and vacuole (each accounting for 7.1%). In addition, phylogenetic analysis showed that TrWRKY79 was clustered in a conserved AtWRKY subfamily, indicating that it had functional similarity with homologous genes involved in stress response.

3.2. Transactivation Activity Assay of TrWRKY79

To determine whether TrWRKY79 has transcriptional activation activity, the full-length TrWRKY79 gene and its truncated fragment were cloned into the pGBKT7(BD) yeast expression vector based on the results of bioprediction from the SMART website (Figure 1a). The resulting recombinant plasmids BD-TrWRKY79, BD-TrWRKY79-N (N-terminal), BD-TrWRKY79-W (WRKY structural domain), and BD-TrWRKY79-C (C-terminal) were transformed into yeast competent cells. Single colonies grown on SD/-Trp solid medium were selected and cultured overnight in 1 mL of SD/-Trp liquid medium. After centrifugation, the yeast cells were resuspended in 0.9% NaCl and spotted onto both SD/-Trp and SD/-Trp/-His media. After five days of incubation, all transformants grew equally well on SD/-Trp medium but displayed differential growth on SD/-Trp/-His medium (Figure 1b). Yeast cells containing the full-length TrWRKY79 or its C-terminal fragment exhibited the most robust growth on SD/-Trp/-His medium, indicating that the C-terminal region likely serves as the core transcriptional activation domain. These results clearly demonstrate that TrWRKY79 has transcriptional activation activity, and only the BD-TrWRKY79-C construct enabled growth on SD/-Trp/-His medium among the truncated variants, further confirming that the transcriptional activation function is localized to the C-terminal domain.

3.3. Overexpression of TrWRKY79 Enhances Cold Stress Tolerance in Transgenic Arabidopsis

The pCAMBIA1300-TrWRKY79 construct was introduced into Agrobacterium tumefaciens and subsequently transformed into Arabidopsis thaliana using the floral dip method. Transgenic seeds were harvested upon maturation and screened on kanamycin-containing medium. A total of ten independent T0 transgenic lines were obtained, and PCR analysis using genomic DNA as the template confirmed the presence of the target gene fragment in all transgenic lines, whereas no amplification was detected in the wild-type (WT) plants (Figure 2a). Homozygous lines were generated from these transformants through further selection. Among them, lines S2, S4, and S8 were selected for subsequent analysis. Total RNA was extracted from these lines and reverse-transcribed into cDNA for qRT-PCR analysis. The results showed that TrWRKY79 expression was undetectable in the WT plants but was clearly expressed in lines S2, S4, and S8, confirming successful gene expression in the transgenic plants (Figure 2b). To further assess the role of TrWRKY79 in cold stress tolerance, potted growth experiments were performed using both the WT and transgenic lines. Four-week-old seedlings were subjected to cold treatment at 4 °C for 24 h. Compared with the WT plants, which exhibited growth retardation and significant leaf wilting, transgenic lines S2, S4, and S8 showed minimal leaf damage under the same conditions, indicating that overexpression of TrWRKY79 from Trifolium repens confers enhanced tolerance to cold stress in Arabidopsis (Figure 2c).

3.4. TrWRKY79 Enhances Physiological Tolerance to Cold Stress in Arabidopsis

To evaluate the role of TrWRKY79 in response to cold stress, transgenic Arabidopsis lines overexpressing the gene (S2, S4, and S8) and wild-type (WT) plants were subjected to low-temperature treatment. Physiological parameters, including chlorophyll (Chl) content, malondialdehyde (MDA) level, proline (Pro) accumulation, and the activities of antioxidant enzymes (CAT, POD, and SOD), were measured. Under normal conditions, no significant differences were observed between the transgenic and WT plants across all parameters. However, following cold stress, the transgenic lines exhibited significantly improved physiological responses. In particular, they maintained significantly higher total chlorophyll content compared to the wild-type plants (p < 0.05, Figure 3a), suggesting a reduced rate of chlorophyll degradation. Although increased chlorophyll levels may contribute to maintaining photosynthetic potential, further biophysical analyses—such as chlorophyll fluorescence or PAM measurements—are required to confirm actual photosynthetic efficiency. Moreover, the transgenic lines accumulated significantly less MDA than the WT plants (p < 0.05, Figure 3b), suggesting lower levels of lipid peroxidation and better preservation of membrane integrity. Proline levels were markedly increased in the transgenic plants (p < 0.05, Figure 3c), reflecting enhanced osmotic adjustment and stress adaptation. Additionally, the activities of CAT, POD, and SOD were significantly higher in the transgenic lines than in the WT plants (p < 0.05, Figure 3d–f), indicating more efficient reactive oxygen species (ROS) scavenging and mitigation of oxidative stress. Collectively, these findings demonstrate that TrWRKY79 plays a critical role in enhancing cold stress tolerance by improving multiple physiological defense mechanisms.

3.5. Binding Site Prediction of WRKY Transcription Factors

To elucidate the potential regulatory mechanism of TrWRKY79 in transgenic Arabidopsis, a genome-wide systematic scan of 2000 bp upstream promoter regions of all genes was performed based on W-box elements. Using the conserved WRKY-binding core motif TTGAC[C/T], tens of thousands of potential binding sites were identified across promoter sequences. For each site, the flanking 5 bp upstream and downstream sequences were extracted, generating a large set of W-box extended motifs ranging from 10 to 15 bp in length. Frequency analysis revealed several high-abundance sequences, including AAACTTGACTCGATT, TTGACTTTATA, and TACATTGACCCAGAA, which retained the canonical WRKY binding core while exhibiting both variability and conservation across different promoter contexts (

Table A3. Extended sequence of W-boxes.

Name	Sequence	Name	Sequence
Wbox1	AAACTTGACTCGATT	Wbox11	GGATTTTGACCACTG
Wbox2	TTGACTTTATA	Wbox12	TTCGATTGACCA
Wbox3	TTGACTATCAC	Wbox13	GAATCTTGACTAG
Wbox4	TACATTGACCCAGAA	Wbox14	CATTGACTCCCAC
Wbox5	TTGACCTCTTC	Wbox15	GGACCTTGACTTGAA
Wbox6	TTTCATTGACTAGTT	Wbox16	TTGACCATTTT
Wbox7	GACGATTGACTGAGA	Wbox17	CTTGTTGACTATACT
Wbox8	GAAGCTTGACTCA	Wbox18	TGCTCTTGACTTTCT
Wbox9	CTGTATTGACTGGCC	Wbox19	ATTAATTGACTAAAA
Wbox10	AGTTGACTATCAC	Wbox20	TTTTGTTGACT

). These high-frequency motifs were formatted in FASTA and used as queries for BLAST searches against the Arabidopsis 2000 bp promoter database to predict candidate TrWRKY79 target genes. The BLAST results showed that many of these extended W-box motifs were enriched in the promoter regions of genes involved in plant growth, development, and abiotic stress responses, suggesting that TrWRKY79 may regulate downstream gene expression via these cis-elements in the transgenic background. To further validate the potential protein–DNA interactions, we selected the top 100 nucleotide sequences containing W-box elements from the BLAST search results, based on the lowest E-values and supported by literature evidence. These sequences represent high-confidence candidate binding sites. Subsequently, Protenix was used to predict the protein–DNA complex structures between these sequences and the TrWRKY79 protein. The predicted interactions with an ipTM score above 0.8 are presented in

Table 1. Binding prediction of TrWRKY79 to target sequences.

Gene	Sequences	Reverse Complemented Sequences	ipTM
AT1G35340	GAAATCGAGTCAAGAAAT	ATTTCTTGACTCGATTTC	0.86
AT2G26180	ACTCTTGACTCGATTTTT	AAAAATCGAGTCAAGAGT	0.85
AT1G67770	AAAGAACCTTGACTTGTT	AACAAGTCAAGGTTCTTT	0.85
AT5G17970	GTAAAAGCTTGACTCAGC	GCTGAGTCAAGCTTTTAC	0.85
AT3G51840	TTCTTTGCTTGACCGCTT	AAGCGGTCAAGCAAAGAA	0.84
AT2G40120	GTTTCTTGACTCGATTCT	AGAATCGAGTCAAGAAAC	0.84
AT5G07010	ACTTAAGCTTGATGAATA	TATTCATCAAGCTTAAGT	0.83
AT1G48330	AAAACTTGACCCGATTAA	TTAATCGGGTCAAGTTTT	0.83
AT5G49730	TTTTACTTGACTCTTCTA	TAGAAGAGTCAAGTAAAA	0.82
AT1G20440	AGAGTGACCACCCCAATG	CATTGGGGTGGTCACTCT	0.82
AT2G39350	GATAACTTGACTCGAACC	GGTTCGAGTCAAGTTATC	0.82
AT3G19090	GATAACTTGACTCGTTTG	CAAACGAGTCAAGTTATC	0.82
AT1G09530	ATTAAACTTGTCTCGATA	TATCGAGACAAGTTTAAT	0.82
AT3G59150	TTTGTTTGACTGAAATTT	AAATTTCAGTCAAACAAA	0.81
AT1G69020	GACACCTTGACTTGATGT	ACATCAAGTCAAGGTGTC	0.81
AT2G04630	CTGAACTTGACTCGAAAT	ATTTCGAGTCAAGTTCAG	0.81
AT2G30910	ATGAAGCTTGACTCAATC	GATTGAGTCAAGCTTCAT	0.81
AT3G45525	TTACATTTTGACCACTGT	ACAGTGGTCAAAATGTAA	0.81
AT5G13320	AAAACATTGACCCAGACT	AGTCTGGGTCAATGTTTT	0.81
AT5G23903	GATACCTTGACTTGACGG	CCGTCAAGTCAAGGTATC	0.81
AT3G05220	AAATTATTGACCATATTA	TAATATGGTCAATAATTT	0.81
AT5G47910	AAGGATTTTGACCAGACC	GGTCTGGTCAAAATCCTT	0.81
AT2G36270	CAAACTTTGACTATTTCT	AGAAATAGTCAAAGTTTG	0.80
AT1G20450	AGTCCGATTGGCCCACAT	ATGTGGGCCAATCGGACT	0.80
AT2G42540	ATGTACTTGACGAGATCG	CGATCTCGTCAAGTACAT	0.80
AT1G43890	CTTTTGACTAATTAGTTT	AAACTAATTAGTCAAAAG	0.80
AT4G03165	AAAGAATCTGGACTGAAA	TTTCAGTCCAGATTCTTT	0.80
AT1G75700	GATTGTTGACTATTTAAA	TTTAAATAGTCAACAATC	0.80
AT4G13730	GTTTGCTTGTCTCAACTT	AAGTTGAGACAAGCAAAC	0.80
AT3G58130	TTTAACTTAACTCGTGAA	TTCACGAGTTAAGTTAAA	0.80
AT2G14920	TTTACATTGACCCCAGCT	AGCTGGGGTCAATGTAAA	0.80
AT1G20180	TGATACATTGACCCATTC	GAATGGGTCAATGTATCA	0.80
AT2G25905	ATGGACCTTAACTAGAAG	CTTCTAGTTAAGGTCCAT	0.80
AT2G44140	CGTAACTTGACTCGATAT	ATATCGAGTCAAGTTACG	0.80
AT3G52900	ACGGATTTTGACCTCTCA	TGAGAGGTCAAAATCCGT	0.80
AT3G61710	CTACGGAAACTTGACTCG	CGAGTCAAGTTTCCGTAG	0.80
AT4G33390	TGGAAGCTTGGCTCATAT	ATATGAGCCAAGCTTCCA	0.80

, while those with scores below 0.8 are listed in

Table A4. The prediction result A5 of iPTM below 0.8.

Gene	Sequences	Reverse Complemented Sequences	ipTM
AT5G51140	TTAGATTGACAGCGACCA	TGGTCGCTGTCAATCTAA	0.79
AT5G48953	AGTACTTTGACCCAGATA	TATCTGGGTCAAAGTACT	0.79
AT2G07680	TTGGATCCTTGACTTGAA	TTCAAGTCAAGGATCCAA	0.79
AT2G30050	ATAACTTTGACCCAGATC	GATCTGGGTCAAAGTTAT	0.79
AT2G38740	TAGGATTTTGACCACAAG	CTTGTGGTCAAAATCCTA	0.79
AT3G53810	ATGGATTTTGACCAGTTG	CAACTGGTCAAAATCCAT	0.79
AT3G58890	GAAGACCTTGACTTGTAC	GTACAAGTCAAGGTCTTC	0.79
AT1G58602	TTCGTAGCTTGACTCATA	TATGAGTCAAGCTACGAA	0.78
AT1G26200	TTGAGGAATCTTGACTAG	CTAGTCAAGATTCCTCAA	0.78
AT3G19663	TGGGACCATGACTTGTTC	GAACAAGTCATGGTCCCA	0.78
AT3G42940	ATAGATTTAGACCACTGT	ACAGTGGTCTAAATCTAT	0.78
AT5G67580	GCTTCTTGACCTGCAATG	CATTGCAGGTCAAGAAGC	0.78
AT1G70944	AGTGGATTTTGACCATGT	ACATGGTCAAAATCCACT	0.78
AT3G25855	ATCCATGACACGATTTTG	CAAAATCGTGTCATGGAT	0.77
AT2G34630	CCCCCCTTGACTCCATCC	GGATGGAGTCAAGGGGGG	0.77
AT4G15530	GAGAAACTATCACCGATC	GATCGGTGATAGTTTCTC	0.77
AT5G22340	TCAAGTTGACCAACAAAT	ATTTGTTGGTCAACTTGA	0.77
AT4G24380	GTGGATTTGACCAGACAT	ATGTCTGGTCAAATCCAC	0.77
AT3G29020	AAAATGACCTTGAAAATT	AATTTTCAAGGTCATTTT	0.77
AT1G70560	AAAGGTTTTTGACCACGT	ACGTGGTCAAAAACCTTT	0.77
AT1G16770	TGTTACATAGACCCAGAG	CTCTGGGTCTATGTAACA	0.77
AT5G06790	CTTGACCTTAACTTGAAG	CTTCAAGTTAAGGTCAAG	0.77
AT5G46295	TTACCTTGACTTGATCTT	AAGATCAAGTCAAGGTAA	0.77
AT3G50700	GTGACCGACTTGAATACC	GGTATTCAAGTCGGTCAC	0.76
AT1G14770	TATCATAGCTTGACTTCT	AGAAGTCAAGCTATGATA	0.76
AT4G00120	CATGATTTTGACCACACA	TGTGTGGTCAAAATCATG	0.76
AT1G55310	AGATTGAAGCTTGACTCA	TGAGTCAAGCTTCAATCT	0.76
AT2G03130	TTATACATTGTCCCAGCC	GGCTGGGACAATGTATAA	0.76
AT2G21830	GAGACCTTGTCTTGAAGC	GCTTCAAGACAAGGTCTC	0.76
AT3G20210	TGTTACATTGACCCTGAG	CTCAGGGTCAATGTAACA	0.76
AT5G06580	TGACTACAGTGACCCAGA	TCTGGGTCACTGTAGTCA	0.76
AT1G45403	TGTTGCGAAGCTTGTCCA	TGGACAAGCTTCGCAACA	0.75
AT4G23100	CGTCAAGCTTGACGAATT	AATTCGTCAAGCTTGACG	0.75
AT2G33010	AAAAAACTATCACTCTCT	AGAGAGTGATAGTTTTTT	0.75
AT3G08820	GACCTTGCTGTATATGTA	TACATATACAGCAAGGTC	0.75
AT2G39210	GTTTGGACCTTGACTGAT	ATCAGTCAAGGTCCAAAC	0.75
AT3G25014	CTCGGATTTTGTCCACTA	TAGTGGACAAAATCCGAG	0.75
AT1G43090	AGAAGGACCTTGACTGAC	GTCAGTCAAGGTCCTTCT	0.75
AT3G16110	AATGGTTGACTATTGAAT	ATTCAATAGTCAACCATT	0.74
AT2G30432	TGACTTTGACCCACACAT	ATGTGTGGGTCAAAGTCA	0.74
AT5G51750	CACTTTTGACCAATGAAA	TTTCATTGGTCAAAAGTG	0.74
AT3G18370	CGTCACTTTGACCCAGAA	TTCTGGGTCAAAGTGACG	0.74
AT3G23123	ACTTTTTTGACCACTGTG	CACAGTGGTCAAAAAAGT	0.74
AT1G43100	AGAAGGACCTTGACTGAC	GTCAGTCAAGGTCCTTCT	0.74
AT4G08878	TAATCTGTTCGATTGACC	GGTCAATCGAACAGATTA	0.74
AT2G02870	CCTTTTGACCACTGAAAT	ATTTCAGTGGTCAAAAGG	0.73
AT2G47160	TTGACGATTTGACTGAGA	TCTCAGTCAAATCGTCAA	0.73
AT4G34940	ACAGATTTTGTCCACTGA	TCAGTGGACAAAATCTGT	0.73
AT5G45310	AAAACGATTGACTGATTA	TAATCAGTCAATCGTTTT	0.73
AT5G59890	CATGAGGACCTTGCTGCA	TGCAGCAAGGTCCTCATG	0.73
AT5G22560	AAAATGAAGCTTGACACA	TGTGTCAAGCTTCATTTT	0.73
AT3G25011	TCGGATTTTGTCCACTAG	CTAGTGGACAAAATCCGA	0.72
AT5G19640	AATTGGACCTTGACTCAA	TTGAGTCAAGGTCCAATT	0.72
AT5G17167	GGCACTTGACTCGGTTGC	GCAACCGAGTCAAGTGCC	0.72
AT5G11350	CAATTCGATTGACCAATG	CATTGGTCAATCGAATTG	0.71
AT3G56270	CTCCATTGACCCAGAGAA	TTCTCTGGGTCAATGGAG	0.71
AT4G26270	CTTGACCTTGACTTGATA	TATCAAGTCAAGGTCAAG	0.71
AT2G14080	TTACATTGTCCCAGAGCA	TGCTCTGGGACAATGTAA	0.70
AT3G51530	CCACGGTTGACTGAGAAG	CTTCTCAGTCAACCGTGG	0.70
AT4G23130	AGTTGACCTTGACTTGCA	TGCAAGTCAAGGTCAACT	0.70
AT4G34930	CAGATTTTGTCCACTGAT	ATCAGTGGACAAAATCTG	0.70
AT3G57760	TTCGATTGACCAAAATCA	TGATTTTGGTCAATCGAA	0.70
AT4G28150	AGTCATTGACCCAGTTTC	GAAACTGGGTCAATGACT	0.70

. The ipTM score (interchain predicted Template Modeling score) is a metric used to estimate the confidence of protein–DNA or protein–protein interaction interfaces, with higher values indicating greater prediction reliability [34]. High-confidence structural models were visualized using ChimeraX, with key DNA-binding interfaces illustrated in Figure 4. Collectively, these results support a potential regulatory role of TrWRKY79 through stable interaction with specific W-box extended sequences, highlighting its capacity for transcriptional modulation in a heterologous system.

3.6. Expression Analysis of Resistance-Related Genes in Arabidopsis thaliana Transgenic for TrWRKY79 at Low Temperature

WRKY transcription factors function as essential regulators in plant stress responses by specifically recognizing W-box cis-acting elements in the promoters of target genes, thereby modulating downstream gene expression and influencing various physiological processes. To elucidate the regulatory role of TrWRKY79 under cold stress, transgenic Arabidopsis lines overexpressing TrWRKY79 were bred and subjected to 24 h of cold treatment at 4 °C together with wild-type (WT) plants. Based on prior promoter element prediction analyses (Section 2.6), several key cold-responsive genes were selected for expression analysis. The quantitative real-time PCR (qRT-PCR) results shown in Figure 5 indicate that the expression levels of several cold-responsive genes were significantly upregulated in the transgenic lines, including AT1G20450 (AtERD10), AT1G20440 (AtCOR47), AT2G36270 (AtABI5), AT1G43890 (AtRAB18), and AT2G42540 (AtCOR15A). Among them, AtCOR15A exhibited the most substantial increase, suggesting it may be a direct transcriptional target of TrWRKY79. The elevated expression of these genes likely contributes to enhanced freezing tolerance and improved cellular protection mechanisms in the transgenic plants. Collectively, these findings indicate that TrWRKY79 acts as a critical upstream transcriptional regulator under cold stress conditions, orchestrating the activation of multiple genes involved in cold acclimation pathways.

4. Discussion

WRKY transcription factors are a prominent class of regulators in plants, extensively involved in modulating responses to both biotic and abiotic stresses. These factors can function as either positive or negative regulators of transcription. For instance, several WRKY proteins, such as AtWRKY18, are known transcriptional activators, while others, like AtWRKY40, OsWRKY51, and OsWRKY71, act as repressors [35,36,37,38]. Interestingly, some WRKY genes exhibit dual functionality. For example, AtWRKY6 can promote gene expression related to senescence and defense responses while also negatively regulating its own promoter activity [39]. In our study, the C-terminal region of TrWRKY79 exhibited strong transcriptional activation capacity (Figure 1), consistent with the typical feature of activation domains being localized to the C-terminal region of WRKY family members [40]. This implies that TrWRKY79 may exert its regulatory function by activating stress-responsive downstream genes.

With the advancement of biotechnology, the use of transgenic approaches to enhance crop stress resistance has become increasingly practical. Although the involvement of WRKY family proteins in abiotic stress responses is well documented in model plants, studies focusing on WRKY genes in Trifolium repens remain limited. In this study, we cloned the TrWRKY79 gene from T. repens and analyzed its function through heterologous expression in Arabidopsis thaliana. Under low temperature stress, TrWRKY79-overexpressing Arabidopsis plants showed significantly improved cold tolerance compared to wild-type plants (Figure 2c). Physiological assessments revealed that transgenic lines maintained higher chlorophyll content under cold stress, suggesting that TrWRKY79 may help sustain photosynthetic efficiency, possibly by modulating genes involved in light harvesting, such as CAB or LHCB (Figure 3a). Additionally, the increased proline accumulation and decreased malondialdehyde (MDA) levels in transgenic plants indicate improved osmotic adjustment and membrane stability (Figure 3b,c). The significantly enhanced activities of antioxidant enzymes (CAT, POD, SOD) suggest that TrWRKY79 may activate ROS-scavenging pathways, thereby improving cellular protection under oxidative stress (Figure 3d–f). Notably, the above physiological indices detected in transgenic TrWRKY79 Arabidopsis under cold stress were similar to the results of transgenic VhWRKY44 Arabidopsis [26]. Collectively, these findings support the notion that TrWRKY79 positively regulates cold and salt stress tolerance without compromising normal growth, making it a promising candidate for genetic improvement in stress resistance in crops.

WRKY transcription factors typically regulate gene expression by recognizing W-box cis-elements in the promoter regions of target genes [41]. In this study, we identified numerous extended W-box motifs through genome-wide promoter scanning and sequence analysis in Arabidopsis. Protein–DNA binding predictions using Protenix further suggested a specific interaction between TrWRKY79 and W-box-containing sequences (Figure 4). Functional assays showed that overexpression of TrWRKY79 significantly upregulated several well-characterized cold-responsive genes, including AtCOR47, AtABI5, AtRAB18, AtCOR15A, and AtERD10, thereby enhancing the cold stress signaling network in transgenic plants (Figure 5). These findings are consistent with previous reports on the involvement of WRKYs in cold response, such as the role of AtWRKY34 in pollen cold tolerance and the positive regulation of rice cold response by OsWRKY76 [20,42]. As a member of the WRKY family, TrWRKY79 likely contributes to an enhancement in cold tolerance by activating a suite of downstream genes involved in stress adaptation (Figure 6). Our expression analysis suggests that TrWRKY79 may be involved in both ABA-dependent and ABA-independent responses under cold stress, as its overexpression led to the upregulation of both ABA-related (ABI5) and cold-responsive (COR47, COR15A, ERD10) genes (Figure 5). Previous studies have shown that WRKY transcription factors can participate in parallel signaling cascades to coordinate abiotic stress responses, including direct activation of COR genes and interaction with ABA signaling components, such as ABI5 [43,44]. Although our data support a correlation with these pathways, further experiments—such as genetic analysis or ABA treatment—will be required to validate the functional roles of TrWRKY79 in these regulatory networks. Therefore, the proposed model (Figure 6) is intended as a conceptual framework for hypothesis generation rather than a confirmed regulatory mechanism. These two pathways synergize to enhance plant tolerance under cold stress. However, while this study elucidates the function of TrWRKY79 in Arabidopsis, its endogenous targets and regulatory mechanisms in white clover remain to be fully characterized. Future studies should aim to dissect its native regulatory network and functional roles in white clover under cold stress.

5. Conclusions

This study systematically elucidates the biological function and molecular mechanism of the TrWRKY79 transcription factor from Trifolium repens in response to low-temperature stress. Cloning and structural prediction revealed that TrWRKY79 encodes an unstable, nucleus-localized protein with a typical WRKY domain and strong transcriptional activation activity. Overexpression of TrWRKY79 in Arabidopsis thaliana significantly enhanced cold tolerance, as evidenced by improved photosynthetic performance, reduced membrane damage, enhanced osmotic adjustment, and increased antioxidant enzyme activities under cold stress. At the molecular level, TrWRKY79 directly or indirectly activated key cold-responsive genes—such as AtCOR15A, AtABI5, and AtERD10—by binding to specific W-box cis-elements within their promoter regions. Protein–DNA docking simulations further supported the specific interaction between TrWRKY79 and extended W-box motifs, indicating its potential regulatory role in the transcriptional cold-response network. Collectively, these findings uncover the molecular mechanism underlying TrWRKY79-mediated cold tolerance and provide new insights into the WRKY gene family in white clover, offering valuable genetic resources for molecular breeding of cold-tolerant forage crops. In the future, we will characterize TrWRKY79 function in white clover under cold stress through transgenic and gene-editing approaches, which will be helpful for forage crop genetic breeding, especially in cold tolerance.