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Genome-wide identification, characterization and expression analysis of WRKY transcription factors under abiotic stresses in Carthamus tinctorius L

BMC Plant Biology volume 25, Article number: 81 (2025) Cite this article

Abstract

Background

WRKY transcription factors constitute one of the largest families of plant transcriptional regulators, playing pivotal roles in plant responses to biotic and abiotic stresses, as well as in hormonal signaling and secondary metabolism regulation. However, a comprehensive analysis of the WRKY family in Carthamus tinctorius (safflower) is lacking. This study aims to identify and characterize WRKY genes in safflower to enhance understanding of their roles in stress responses and metabolic regulation. Safflower, valued for its ornamental, medicinal, and culinary uses, exhibits significant resilience to salt, alkali, and drought. By elucidating the functions and expression patterns of WRKY genes, we aim to enhance breeding strategies for improved stress tolerance and metabolic traits in crops.

Results

In this study, we identified 84 WRKY genes within the safflower genome, and classified them into three primary groups (Groups I, II, and III) based on molecular structure and phylogenetic relationships. Group II was further subdivided into five subgroups (II-a, II-b, II-c, II-d, and II-e). Gene structure, conserved domain, motif, cis-elements, and expression profiling were performed. Syntenic analysis revealed that there were 27 pairs of repetitive fragments. Expression profiles of CtWRKY genes were assessed across diverse tissues, colored cultivars, and abiotic stresses such as ABA, drought, and cold conditions. Several CtWRKY genes (e.g., CtWRKY44, CtWRKY63, CtWRKY65, CtWRKY70 and CtWRKY72) exhibited distinct expression patterns in response to cold stress and during different developmental stages. Additionally, CtWRKY13, CtWRKY69, CtWRKY29, CtWRKY56, and CtWRKY36 were upregulated across different flower colors. The expression patterns of CtWRKY48, CtWRKY58, and CtWRKY70 varied among safflower cultivars and flower colors. After exposure to drought stress, the expression levels of CtWRKY29 and CtWRKY58 were downregulated, while those of CtWRKY56 and CtWRKY62 were upregulated.

Conclusion

This study identified 84 WRKY genes in Carthamus tinctorius and classified them into three groups, with detailed analyses of their structure, conserved domains, motifs, and expression profiles under various stresses. Notably, several WRKY genes such as CtWRKY44, CtWRKY63, and CtWRKY72 displayed significant expression changes under cold stress, while CtWRKY56 and CtWRKY62 were responsive to drought stress. These findings highlight the critical roles of specific WRKY genes in abiotic stress tolerance and developmental regulation in safflower.

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Backgrounds

Carthamus tinctorius L., commonly known as false saffron, is a valuable plant with significant economic and medicinal importance. It belongs to the Asteraceae family and is primarily found in arid regions across various countries. With over 4000 years of cultivation, C. tinctorius is renowned for its flower petals, which produce a diverse spectrum of colors, making them highly valuable in both the culinary and textile industries [1, 2]. In addition to its historical use as a natural dye, C. tinctorius is primarily cultivated for its seed oil, which is rich in essential fatty acids and bioactive compounds, including flavonoids and phenylethanoid glycosides [2]. These compounds not only contribute to its medicinal value but also to its importance as a nutraceutical. The plant's diverse flower colors are attributed to anthocyanins, further highlighting its relevance in agricultural and industrial applications [3, 4].

Recent research has focused on the genetic regulatory networks involved in the complex process of flowering in C. tinctorius. Among the transcriptional regulators, the WRKY gene family has emerged as a key player, regulating vital plant processes and signaling networks [5]. The first WRKY protein, SPF1 from Ipomoea batatas, laid the foundation for understanding this gene family [6]. Subsequent studies have identified numerous WRKY genes across various plant species, such as Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa, Petroselinum crispum, and Panax ginseng, with a particular focus on their roles in secondary metabolite production in medicinal plants. Analysis of sequence similarities has revealed a substantial number of WRKY genes in several species, Notably, 57, 61, 62, 72, 85, 90, 94, 98, 102, 103 and 239 WRKY genes have been identified in Cucumis Melo L. [7], Cucumis sativus L. [8], Capsicum annuum [9], A. thaliana [10], Vigna angularis and Vigna radiata [11], H. annuus [12], Sorghum bicolor (L.) moench [13], O. sativa japonica and O sativa indica [14], Pyrus bretschneideri [15], and Gossypium hirsutum [16], respectively.

The WRKY proteins are composed of a highly conserved WRKY domain (approximately 60 amino acids long). This domain is characterized by a distinct WRKYGQK sequence at the N-terminus and a zinc finger motif (C2H2 or C2HC) at the C-terminus [17]. Plant-specific WRKY transcription factors (TFs) are defined by one or two conserved WRKY domains located in the N-terminus, typically followed by a zinc finger motif in the C-terminus. WRKYGEK and WRKYGKK heptapeptides are examples of such conserved domains. These domains collectively constitute the WRKY DNA-binding domain, which plays a critical role in DNA binding. WRKY TFs specifically bind to cis-acting elements known as W-boxes (TTGACC/T), located in the promoters of target genes [18], thereby modulating their expression. Indeed, the zinc finger-like motif present in WRKY TFs holds great importance in plant evolution [18].

Based on the number of WRKY domains and the structure of zinc finger motifs, WRKY proteins can be classified into three major groups: Group I, characterized by two domains and a single C2H2 zinc finger; groups II and III, comprising one domain with a C2H2 zinc finger and a C2HC zinc finger, respectively [19]. Group I can be further divided into two subgroups, Ia and Ib, based on the presence of two WRKY domains with C2H2 zinc finger motifs (C-X4-5-C-X22-23-H-X1-H) located at the N-terminus and C-terminus, respectively [20]. Group II can be subdivided into five subgroups (IIa-IIe) based on their primary amino acid sequences, comprising proteins with one WRKY domain and a C2H2 zinc finger motif, while Group III can be categorized into subgroups IIIa and IIIb, which include proteins with one WRKY domain and one C2HC zinc finger motif [19]. These TFs, including WRKY proteins, play a critical role in the activation, repression, and modulation of transcription, both independently and in collaboration with other regulatory proteins. Notably, they are integral to signal transduction pathways and the regulation of gene expression, enabling crops to adapt effectively to a range of environmental challenges [21]. Extensive research has demonstrated the involvement of WRKY TFs in various physiological processes, highlighting their significant contributions to plant growth and development.

Despite extensive research on the importance of WRKY genes in various plants, their specific roles in C. tinctorius, particularly in relation to flavonoid biosynthesis, remain largely unexplored. To address this gap, the present study undertakes a comprehensive genome-wide identification, characterization, and expression analysis of WRKY TFs in C. tinctorius. We systematically analyzed the C. tinctorius WRKY (CtWRKY) genes using the whole-genome database and investigated their phylogenetic relationships, chromosomal distribution, conserved motifs, and potential protein interactions. Additionally, we examined the expression patterns of CtWRKY genes across different tissues and cultivars through RNA sequencing. The findings of this research aim to provide deeper insights into the regulatory functions of WRKY genes in C. tinctorius, particularly in relation to metabolic pathways such as flavonoid biosynthesis.

Results

Genome-wide identification of CtWRKY genes

The protein sequences of C. tinctorius were analyzed using HMMER version 3.0 [22], with an E-value threshold of < 1e−5. Subsequently, the candidate sequences were subjected to further filtration using Pfam [23], NCBI conserved domains [24], and SMART database [25] to confirm the presence of complete WRKY domains. A total of 84 CtWRKY genes were successfully identified and designated as CtWRKY1 to CtWRKY84, based on their respective positions on the C. tinctorius chromosomes (Table S1).

Comprehensive information on the CtWRKY genes, including gene names, IDs, positions (start and end), protein lengths, molecular weights, theoretical pI, instability index, and subcellular localization, are presented in Table S1. The lengths of CtWRKY proteins varied, with the shortest being CtWRKY28 at 159 amino acids and the longest being CtWRKY70 at 745 amino acids, yielding an average protein length of approximately 355 amino acids. Their molecular weights also varied significantly; CtWRKY28 had a molecular weight of 17,551.09 Da, while CtWRKY70 weighed 83,840.87 Da, with an average molecular weight of around 39,517.42 Da. Isoelectric points were determined using ExPASy [26], revealing the lowest pI at 4.83 for CtWRKY63 and the highest at 10.15 for CtWRKY35. Notably, 53.48% of CtWRKY proteins had isoelectric points below 7, indicating a tendency towards acidity. The instability index ranged from 36.62 for CtWRKY80 to 69.74 for CtWRKY3, with only three proteins exhibiting indices below 40, suggesting that most CtWRKY proteins are relatively stable (Supplementary Table S1).

Evolutionary classification and phylogenetic analysis of CtWRKY gene family

To investigate the evolutionary relationships between CtWRKY and AtWRKY proteins, a multiple sequence alignment and phylogenetic tree were constructed using the neighbor-joining method (Fig. 1). The CtWRKY genes were classified into seven distinct subgroups based on their Arabidopsis thaliana counterparts (Figs. 1 and 2). Subgroup III contained the largest number of genes in C. tinctorius, comprising 24 members, followed by subgroup I with 15 genes, subgroup IIc with 14 genes, subgroup IIe with 12 genes, subgroup IIb with 8 genes, and subgroup IIa with 6 genes. Subgroup IId had the fewest members, totaling 5 genes (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree and conserved motif analysis of CtWRKY. A The seven subgroups are marked in different colors on the periphery of the circle. Red dots indicate safflower CtWRKY proteins. The phylogenetic tree was constructed using MEGA6, with bootstrap values based on 1,000 iterations. B Multiple alignment analysis of CtWRKY proteins

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Interestingly, C. tinctorius and A. thaliana shared an equal number of genes in subgroup IIb. The distribution of A. thaliana genes across subgroups was as follows: subgroup I (14 genes), subgroup III (13 genes), subgroup IIc (12 genes), subgroup IIe (8 genes), subgroup IId (7 genes), and subgroup IIa (3 genes). The most significant disparity was observed in subgroup III, where the number of CtWRKY members was double that of their Arabidopsis counterparts (Fig. 1).

Gene structure and conserved motif analysis

The gene structures of C. tinctorius and A. thaliana WRKY genes were analyzed using TBtools [8] (Fig. 2). Within each subgroup, CtWRKY genes exhibited high structural similarities. Analysis via MEGA version 6 and TBtools revealed distinct patterns in the distribution of untranslated regions (UTRs) at the 3' end of genes, particularly in subgroups IIa and IIb. Subgroups I, IIa, IIb, IIc, IId, and IIe contained 2–6, 2–5, 3–4, 1–4, 2, and 1–3 introns, respectively (Fig. 2). Subgroup III displayed a wider range of intron numbers, with most members containing 2–4 introns. Among the identified WRKY genes, CtWRKY6 had the fewest exons (n = 2), while CtWRKY70 had a highly complex structure with 10 exons (Fig. 2).

Conserved motifs in CtWRKY proteins were analyzed using MEME tools version 5.1.1 [27] (Fig. 2). This analysis revealed ten conserved motifs, with lengths up to 745 amino acids. Each CtWRKY protein exhibited a unique combination of these motifs, indicating specific patterns within particular subgroups. Notably, Motif 8 was the largest in size and the most prevalent in subgroup I (Fig. 2). Some motifs were preferentially distributed across specific subgroups. For instance, Motif 7 was prominently located in the N-terminus of proteins in subgroups IIa, IIb, and III. Subgroup IIa displayed a characteristic motif pattern of 5’−7–1–3-(4)−2–10-3’, except for CtWRKY7, which followed a 5’−1–3–4–2–10–3’ pattern, and CtWRKY8, which had the pattern 5’−7–1–3–2–10–3’ (Fig. 2). Subgroup IIb had a distinct pattern of 5’−7–5-4–3-1–2–10–3’, with CtWRKY22 as the only member starting with Motif 8; the others began with Motif 7 (Fig. 2). Subgroup III exhibited a pattern of 5’−7–9-(6)−1-(3)−4–2–3’, where Motif 7 was predominant in 91.67% of its members (Fig. 2).

In contrast, Motif 5 was primarily found in subgroups I, IIa, IIb, and IIc. Subgroup I featured Motif 1 as the primary motif in 86.67% of its members, although variations were noted in CtWRKY1 and CtWRKY67 (Fig. 2). Subgroup IIb again exhibited a distinct pattern of 5’−7–5-4–3-1–2–10–3’, with the same observations regarding Motif 8 (Fig. 2). Subgroup IIc consistently presented Motif 5 first, except for CtWRKY11, which began with Motif 7. Subgroup IId showed a diverse motif pattern, with 40% of its members starting with Motif 7, another 40% with Motif 1, and one member with Motif 6. The identified pattern for subgroup IId was 5'−7–6-1–3–4–2–3'. In subgroup IIe, the majority (83.33%) started with Motif 6, while subgroups IId and IIe shared a similar pattern of 5'-(6)−1–3–4–2–3' (Fig. 2). Additionally, Motif 9 was exclusively observed in subgroup III (Fig. 2). Overall, Motif 1 represents the WRKY domains in the CtWRKY genes [28].

Fig. 2
figure 2

Gene structure and conserved motif analysis of CtWRKY gene family. The seven subgroups are marked with different background colors. Conserved motifs are represented by different colored boxes

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Gene duplication is known to contribute to gene family expansion and the emergence of novel functions. Our analysis of the C. tinctorius genome identified 27 gene pairs among CtWRKY genes (Supplementary Table S2). Four pairs resulted from tandem duplications, including CtWRKY8 and CtWRKY9, CtWRKY48 and CtWRKY49, CtWRKY63 and CtWRKY64, and CtWRKY69, CtWRKY70, and CtWRKY71. These tandem duplicates were classified into the same subfamily.

To explore the evolutionary relationships among WRKY TFs from different species, a collinearity plot was constructed between C. tinctorius and three other species: A. thaliana, O. sativa, and H. annuus (Fig. 3B). The collinear analysis identified 61 orthologs between C. tinctorius and A. thaliana, 11 orthologs between C. tinctorius and O. sativa, and 128 orthologs between C. tinctorius and H. annuus (Fig. 3C). These findings imply a significant evolutionary distance. Furthermore, the prevalence of one-to-many matches in the collinear relationships indicates that CtWRKY genes display a relatively conserved nature, suggesting that these collinear CtWRKY genes across different species likely share a common ancestral origin.

Fig. 3
figure 3

Chromosomal locations and collinearity analysis of the CtWRKY gene family. A CtWRKY genes are maped on chromosomes; scale bar on the left indicates length of safflower chromosome (Mb). B Collinearity analysis of CtWRKY genes; circle plot was constructed, and the identified collinear genes are linked by colored lines. C Collinearity relationship of CtWRKY genes among A. thaliana, H. annuus, and O. sativa. Identified collinear genes are linked by red lines

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Protein interaction network

Figure 4 depicts the protein interaction network involving CtWRKY TFs. In this network, CtWRKY TFs are represented by red circles, including CtWRKY2, CtWRKY13, CtWRKY15, CtWRKY21, CtWRKY23, CtWRKY27, CtWRKY29, CtWRKY38, CtWRKY41, CtWRKY42, CtWRKY46, CtWRKY53, CtWRKY55, CtWRKY58, CtWRKY59, CtWRKY62, CtWRKY66, CtWRKY72, CtWRKY78, CtWRKY80, and CtWRKY81. The green circles represent key enzymes actively that play significant roles in plant regulatory pathways, particularly highlighting the Mitogen-Activated Protein Kinase (MAPK) signaling pathway (Fig. 4). The biological processes, cellular locations and molecular functions of CtWRKY genes are depicted in Supplementary Fig. 1.

Fig. 4
figure 4

Protein interaction network of CtWRKY from the perspective of A. thaliana homologous genes. Red colour represents CtWRKY transcription factors, and green colour represents key synthase genes. The thicker the gray connecting line, the stronger is the predicted interaction between the two proteins. Note: MPK4 (Mitogen-activated protein kinase 4), MPK3 (Mitogen-activated protein kinase 3), ACS6 (1-aminocyclopropane-1-carboxylate synthase 6), SIB1 (Sigma factor binding protein 1), NPR1(Nonexpresser of PR genes 1), SIB2 (Sigma factor binding protein 2), AT1G18a (Autophagy-related protein 18a), GUN5 (Genomes uncoupled 5), MKS1 (MAP Kinase Substrate 1), and TTG1 (Transparent Testa Glabra1)

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Cis-element analysis of the CtWRKY gene family

To identify specific cis-elements associated with plant growth and development, hormone response, and stress response, an extensive cis-element analysis was performed by retrieving the upstream promoter sequences of the CtWRKY genes from the PlantCARE database (Fig. 5). Notably, all 84 CtWRKY genes were found to harbor at least one type of cis-element. In terms of plant growth and development, specific cis-elements, primarily light-responsive elements such as G-box, Box 4, and GT1-motif, were predominantly enriched (Supplementary Table S3). Additionally, hematopoietic development elements, including the GATA-motif, were identified. The TCT motif, known to be essential for the transcription of ribosomal protein gene promoters, was frequently detected in CtWRKY genes. Regarding hormone response elements, the CtWRKY genes displayed significant enrichment of the SA-responsive element, notably the TGACG-motif. Furthermore, cis-elements associated with abscisic acid (ABA)-induced gene expression, such as ABRE, were commonly found among the CtWRKY genes. In terms of stress response elements, the most prevalent types were those involved in anaerobic induction (ARE-motif) and wounding stress (WUN-motif) (Fig. 5).

Fig. 5
figure 5

Cis-Acting elements analysis of the promoter regions of CtWRKY Gene Family genes related to plant growth and development, hormone response, and stress response

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Expression profiles of the CtWRKY gene family

Using transcriptome data from safflower flowers of various colors (white-W, yellow-Y, light red-LR, and deep red-DR), we examined the expression patterns of the CtWRKY gene family. The results demonstrated distinct expression profiles, indicating differential expression of CtWRKY genes across the different flower colors. Among the 84 analyzed CtWRKY genes, 11, 16, 17, and 10 genes were upregulated in the W, Y, LR, and DR flower samples, respectively (Fig. 6A). In the W flower samples, the identified genes were CtWRKY2, CtWRKY13, CtWRKY16, CtWRKY27, CtWRKY32, CtWRKY37, CtWRKY59, CtWRKY64, CtWRKY66, CtWRKY69, and CtWRKY75. In the Y flower samples, CtWRKY10, CtWRKY18, CtWRKY31, CtWRKY33, CtWRKY48, CtWRKY53, CtWRKY55, CtWRKY57, CtWRKY58, CtWRKY63, CtWRKY67, CtWRKY70, CtWRKY77, CtWRKY83, CtWRKY79 and CtWRKY82 were identified. The LR flower samples contained CtWRKY3, CtWRKY61, CtWRKY24, CtWRKY39, CtWRKY14, CtWRKY25, CtWRKY71, CtWRKY26, CtWRKY42, CtWRKY1, CtWRKY7, CtWRKY50, CtWRKY12, CtWRKY76, CtWRKY81, CtWRKY38 and CtWRKY49. The DR flower samples included CtWRKY11, CtWRKY19, CtWRKY20, CtWRKY22, CtWRKY30, CtWRKY34, CtWRKY35, CtWRKY36, CtWRKY40 and CtWRKY84 (Fig. 6A).

Analysis of gene expression across various tissues revealed distinctive trends, with 64.3% of the CtWRKY genes exhibiting distinct upregulation patterns (Fig. 6B), suggesting their involvement in specific biological processes. At the 1 day after germination (1DAG) time point, several genes (CtWRKY1, CtWRKY5, CtWRKY11, CtWRKY18, CtWRKY29, CtWRKY38, CtWRKY39, CtWRKY44, CtWRKY53, CtWRKY63, CtWRKY58, and CtWRKY66) demonstrated significant upregulation, indicating their potential roles in critical molecular events during seed germination. At the 5DAG time point, CtWRKY32 and CtWRKY73, were upregulated. At 7DAG, CtWRKY54, CtWRKY62, CtWRKY65, and CtWRKY74 were upregulated. Finally, at 10DAG, a diverse set of genes, including CtWRKY6, CtWRKY20, CtWRKY26, CtWRKY43, CtWRKY45, CtWRKY46, CtWRKY47, CtWRKY57, CtWRKY8, CtWRKY13, CtWRKY33, CtWRKY37, CtWRKY59, CtWRKY84, CtWRKY61, CtWRKY62, CtWRKY78, and CtWRKY80, were upregulated. Notably, CtWRKY62 was upregulated at both the 7DAG and 10DAG time points, suggesting its involvement in later stages of seed germination (Fig. 6B).

At the DAF0 stage, several genes, including CtWRKY13, CtWRKY21, CtWRKY24, CtWRKY35, CtWRKY44, CtWRKY50, CtWRKY56, CtWRKY17, CtWRKY64, CtWRKY71, CtWRKY65, CtWRKY68, CtWRKY72, CtWRKY81, CtWRKY82, CtWRKY83, CtWRKY76, and CtWRKY77, were upregulated (Fig. 6B). CtWRKY44, which was upregulated at DAF0, also showed upregulation at 1DAG. Similarly, CtWRKY65 was upregulated at both DAF0 and 7DAG, while CtWRKY13 was upregulated at both DAF0 and 10DAG (Fig. 6B).

The upregulated genes across all days after germination and DAF0 remained consistent in both LL_DAF10 and LL_DAF20 samples. However, in the LL_DAF10 samples, CtWRKY7, CtWRKY28, CtWRKY36, CtWRKY48, CtWRKY69, and CtWRKY79 showed increased expression. Similarly, in the LL_DAF20 samples, CtWRKY15 and CtWRKY70 were upregulated (Fig. 6B). During the DFS stage, diverse genes, including CtWRKY2, CtWRKY5, CtWRKY9, CtWRKY10, CtWRKY14, CtWRKY22, CtWRKY23, CtWRKY25, CtWRKY29, CtWRKY4, CtWRKY12, CtWRKY19, CtWRKY27, CtWRKY34, CtWRKY41, CtWRKY42, CtWRKY50, CtWRKY56, CtWRKY49, CtWRKY51, CtWRKY52, CtWRKY55, CtWRKY58, CtWRKY72, CtWRKY60, and CtWRKY63, exhibited significant upregulation (Fig. 6B). Notably, CtWRKY5, CtWRKY29, CtWRKY58, and CtWRKY63, which were upregulated at 1DAG, also showed upregulation during the DFS stage. Additionally, CtWRKY56 and CtWRKY72, which were upregulated at DAF0, were also found to be upregulated during the DFS stage (Fig. 6B).

The expression profiles of CtWRKY genes in relation to flower color were generally independent of those observed at different stages of DAG and DAF, including LL_DAF10, LL_DAF20, and DFS (Fig. 6A, B). Comparative analysis between different tissues and cultivars with distinct colors revealed interesting patterns. For instance, CtWRKY13 and CtWRKY69, which were upregulated specifically in W flowers, were also found to be upregulated at the DAF0 and 10DAG time points, respectively. In addition, CtWRKY36, which exhibited upregulation in DR flowers, was also upregulated at the LL_DAF10 stage. CtWRKY48 and CtWRKY70, which were upregulated in Y flowers, demonstrated upregulation in LL_DAF10 and LL_DAF20, respectively. Furthermore, CtWRKY58 and CtWRKY63, upregulated in Y samples, exhibited upregulation at both the 1DAG time point and the DFS stage. CtWRKY29, upregulated in LR samples, also showed increased expression at 1DAG and during the DFS stage. Lastly, CtWRKY56, which was upregulated in LR samples, displayed upregulation at both DAF0 and DFS stages (Fig. 6A, B).

Fig. 6
figure 6

Expression profiles of the CtWRKY gene family. A Heatmap of expression profiles for CtWRKY genes in safflower cultivars with distinct colors. The expression levels are displayed by the color bar. B Expression patterns of CtWRKY genes in different tissues. The right bar represents the normalized values of FPKMs by row scale

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Gene expression patterns of CtWRKY following abiotic stress treatment

The WRKY gene family has been implicated in the regulation of plant responses to abiotic stress. To investigate whether the expression levels of CtWRKY genes are associated with stress responses, C. tinctorius seedlings were subjected to various abiotic stresses and stress-related hormones, including ABA, drought, and cold. The CtWRKY genes with LTR, ABRE and MBS elements were selected and surveyed for their expression levels under cold, ABA and drought stress treatment. We focused on CtWRKY genes containing LTR, ABRE, and MBS elements, assessing their expression levels under these stress treatments. Quantitative PCR (qPCR) analysis was conducted to evaluate the expression levels of these genes at multiple time points post-treatment, and the data obtained were recorded (Fig. 7). The observed expression patterns demonstrated substantial alterations in gene transcription in response to different levels of abiotic stress, suggesting that members of the CtWRKY gene family respond variably to multiple stressors. Under ABA stress, the majority of the 26 analyzed genes exhibited low mean values of relative expression but showed upregulation overall. Notably, CtWRKY2, CtWRKY13, CtWRKY19, CtWRKY41, CtWRKY45, CtWRKY48, and CtWRKY80 exhibited significantly higher mean expression patterns. In contrast, CtWRKY4, CtWRKY5, CtWRKY7, CtWRKY8, CtWRKY23, CtWRKY27, CtWRKY58 and CtWRKY76 were initially upregulated, followed by downregulation after 24 h of ABA treatment. Additionally, CtWRKY12, CtWRKY22, CtWRKY27, CtWRKY36, and CtWRKY69 exhibited significant differential expression levels (Fig. 7A).

Under drought stress conditions, most of the 21 genes analyzed also showed low mean relative expression values and significant upregulation, particularly CtWRKY8 and CtWRKY61, which exhibited significantly higher expression levels compared to the other genes. However, both CtWRKY8 and CtWRKY58 were first upregulated before subsequently declining in expression. Furthermore, CtWRKY4, CtWRKY8, CtWRKY29, CtWRKY43, CtWRKY45, and CtWRKY75 were downregulated or had no obvious regulation patterns after 24 h of drought treatment (Fig. 7B).

During cold stress treatment, a significant proportion (47.22%) of the genes showed high mean relative expression values, with CtWRKY5, CtWRKY11, CtWRKY58, and CtWRKY60 demonstrating a substantial upregulation (Fig. 7C). The expression levels of CtWRKY7 and CtWRKY8 were first increased and then declined. Conversely, CtWRKY20, CtWRKY44, CtWRKY49, CtWRKY50, CtWRKY55, CtWRKY70, CtWRKY63, and CtWRKY72 showed differential expression levels, while CtWRKY34, CtWRKY69, and CtWRKY82 exhibited significant downregulation patterns (Fig. 7C).

An obvious trend emerged when comparing relative expression patterns in response to drought and cold treatments. Specifically, CtWRKY8 had the lowest expression level at the 3-h time point, followed by a noticeable spike at 6 h, after which expression gradually decreased and stabilized at the 12- and 24-h time points (Fig. 7B, C). CtWRKY58, consistently exhibited high mean expression values across all abiotic stress treatments, showing upregulation after ABA and cold treatments but downregulation following drought stress (Fig. 7). These findings highlight the diverse and dynamic nature of gene expression patterns in response to ABA, cold, and drought stress. Taken together, CtWRKY genes, especially CtWRKY8 and CtWRKY58, exhibit unique temporal regulation patterns and complex regulatory mechanisms, suggesting their involvement in specific biological processes during abiotic stress responses.

Fig. 7
figure 7

Relative expression of CtWRKY genes following ABA, drought and cold treatments. A Relative expression of CtWRKY genes following ABA treatment. B Relative expression of CtWRKY genes following drought treatment. C Relative expression of CtWRKY genes following cold treatment. Data represents the mean ± SD of three biological replicates. * p < 0.05, ** p < 0.01

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Subcellular localization of CtWRKY60

Most WRKY TFs are known to localize specifically in the nucleus, where they exhibit transcriptional activation or repression activity. The nuclear localization prediction server WoLF PSORT indicated that all CtWRKY proteins are likely to be localized in the nucleus. To validate this prediction, we constructed a CtWRKY60-GFP fusion protein driven by the CaMV 35S promoter (Fig. 8A). We then conducted a transient expression experiment by infiltrating Nicotiana benthamiana leaves with Agrobacterium. A nucleus marker gene fused to mCherry was co-transformed along with GFP to confirm nuclear localization. In the transformed tobacco epidermis, we observed that the green fluorescent protein signals from CtWRKY60-GFP were specifically localized in the nuclei. In contrast, control protoplasts expressing GFP exhibited a ubiquitous distribution throughout the entire cell (Fig. 8B)

Fig. 8
figure 8

Subcellular localization of CtWRKY60. (A) Schematic illustration of the constructs. (B) Transient infiltration of the 35S::GFP and 35S::CtWRKY60-GFP plasmids into Nicotiana benthamiana leaves via Agrobacterium tumefaciens-mediated transformation

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Discussion

In this study, we conducted a comprehensive analysis of the gene structure, protein motifs, gene duplications, and syntenic relationships within the WRKY family in C. tinctorius. We further investigated the expression patterns of WRKY genes under abiotic stress treatments and examined their subcellular localization.

Genome-wide exploration and evolutionary analysis of WRKY genes in C. tinctorius

C. tinctorius is a plant of considerable economic and medicinal value, presenting significant research potential despite limited genomic and transcriptomic data. The WRKY gene family, prevalent across various plant species, plays crucial roles in regulating essential processes such as growth, development, and stress responses [29]. These TFs, in coordination with plant hormones, maintain a balance between development and resilience, significantly influencing a plant’s ability to manage biotic and abiotic stresses. Recent genome sequencing efforts have enabled the identification and characterization of CtWRKY genes, providing insights into their functions and roles in the plant. This research enhances our understanding of the significance and potential of CtWRKYs in molecular processes and their responses to abiotic stressors. In our analysis, we identified 84 CtWRKY members in the C. tinctorius genome. Although this number is fewer than in some species (e.g., Cucumis Melo L., Cucumis sativus L., Capsicum annuum and A. thaliana) [7,8,9,10], it is greater than in others (e.g., Vigna angularis, Vigna radiata, H. annuus, Sorghum bicolor (L.) moench, O. sativa japonica, O. sativa indica, Pyrus bretschneideri and Gossypium hirsutum) [11,12,13,14,15,16]. C. tinctorius possesses fewer WRKYs than certain species (e.g., Cucumis Melo L., Cucumis sativus L., Capsicum annuum and A. thaliana) [7,8,9,10] but more than others (e.g., Vigna angularis, Vigna radiata, H. annuus, Sorghum bicolor (L.) moench, O. sativa japonica, O. sativa indica, Pyrus bretschneideri and Gossypium hirsutum) [11,12,13,14,15,16]. This suggests that gene duplication events may have occurred during the evolution of the C. tinctorius genome.

Gene duplication events, such as tandem, segmental, and whole-genome duplications, have expanded the WRKY gene family in C. tinctorius, similar to observations in other plant species [10, 12, 14]. These duplications have contributed to the diversification of gene functions and the emergence of large gene families. The CtWRKY family was classified into three groups based on conserved WRKY domains and zinc finger motifs, with Group II being the most prevalent (Figs. 1 and 2). Comparisons with A. thaliana revealed similarities among certain WRKY proteins, offering insights into evolutionary relationships and potential functional similarities of CtWRKY proteins with those in other species [14]. Notably, within the WRKY domain WRKYGQK, CtWRKY57 (subgroup I) exhibits a distinctive mutation in the second amino acid (WCKYGQK), differentiating it from other CtWRKY members. Similarly, CtWRKY44 (subgroup III) shows a mutation in the second amino acid (WSKYGQK) (Fig. 1B). In contrast, CtWRKY34, CtWRKY73, CtWRKY74, and CtWRKY80 (all in subgroup IIc) possess significant mutations in the sixth amino acid of the conserved heptapeptide (WRKYGKK). Additionally, CtWRKY18 displays an intriguing mutation in the sixth amino acid (WRKYGAK) (Fig. 1B), highlighting the remarkable diversity among CtWRKY members in the C. tinctorius genome. This pattern aligns with other species' genomes such as S. lycopersicum, Malus domestica Borkh., and C. annuum, all containing this variant of the WRKY domain. The WRKYGQK domain plays a functionally significant role in binding to cis-elements in the promoter region and interacting with target genes [30]. These unique mutations provide insights into the distinctive properties and evolutionary dynamics of CtWRKY proteins, enhancing our understanding of their roles in molecular processes, particularly in response to abiotic stresses.

Our findings align closely with previous research on flowering plants, identifying four primary WRKY TF lineages: group I + IIc, groups IIa + IIb, group IId, and group IIe [31]. In C. tinctorius, we identified subgroups I + IIc, subgroups IIa + IIb, subgroups IId + IIe, and subgroups III + IIe + IIc (Fig. 2). The phylogenetic tree reveals that subgroup III, subgroup IIc, and subgroup IIe each branch into three distinct clades, converging within the same major branch (Fig. 2). Analysis of conserved motifs within the WRKY gene family identified ten motifs, with Motif 8 being predominant in subgroup I and Motif 7 being significant in the N-terminus of proteins, particularly in subgroups IIa, IIb, and notably, subgroup III (Fig. 2). According to the intron–exon structural analysis, the WRKY gene family in C. tinctorius displays 1–6 introns (Fig. 2), which is comparable to Beta vulgaris L. (1–5) [32] but differing from Pennisetum glaucum (0–17) [33] and other economically important crops such as artichoke (1–12) [34]. Within this gene family, CtWRKY6 has the fewest exons (n = 2), while CtWRKY70 is characterized by a more complex structure with 10 exons (Fig. 2). These findings are consistent with Solanum chacoense, which also contains 84 WRKY genes with one to eight exons [35], but differ from Saccharum spontaneum AP85-441, which has 2–6 exons. The low gene structure diversity of CtWRKY genes suggests specific patterns within sister clades and conserved motifs. Additionally, the gene structures, including intron–exon distribution patterns, are generally group-specific, validating the phylogenetic relationships and indicating potential functional similarities among these subgroups.

Figure 3A illustrates 23 tandem duplicates within the same gene subfamily, forming clusters on chromosomes 3, 8, 10, and 11, along with single tandem duplicates on chromosomes 7 and 12. Notably, chromosome 8 contains a significant cluster (CtWRKY47 to CtWRKY51 and CtWRKY54), while chromosome 10 has 11 tandem duplicates (CtWRKY61 to CtWRKY66 and CtWRKY68 to CtWRKY72). These findings suggest that segmental duplications in these regions have contributed to the expansion of the CtWRKY family. We generated collinearity maps comparing the CtWRKY family with monocots (O. sativa) and dicots (A. thaliana and H. annuus) (Fig. 3B, C). The analysis revealed 61 orthologs between C. tinctorius and A. thaliana, 11 orthologs between C. tinctorius and O. sativa, and 128 orthologs between C. tinctorius and H. annuus (Fig. 3C). The presence of collinear CtWRKYs shared between C. tinctorius and H. annuus suggests a close evolutionary relationship and common ancestral origin. Additionally, the lower number of collinear gene pairs between C. tinctorius and monocots compared to dicots indicates that these gene pairs formed after the divergence of dicot and monocot plants. These findings provide compelling evidence for the evolutionary dynamics and shared genetic heritage among different plant species.

The expression and promoter analysis of WRKY genes provide important clues for their functions

WRKY genes play a crucial role in plant growth, development, hormone, and stress responses, enhancing plant tolerance against both biotic and abiotic stressors [36,37,38,39,40,41,42,43,44,45,46,47,48]. Our study on CtWRKY genes revealed specific cis-elements associated with plant growth, hormone response, and stress response, indicating their active involvement in these processes. Notably, many CtWRKY genes contain light-responsive, abscisic acid, and adenylate-uridylate-rich elements, highlighting their versatile roles in hormone regulation and stress responses (Fig. 5). Protein interaction network analysis demonstrated that CtWRKY proteins engage with key enzymes in plant regulatory pathways, particularly the MAPK signaling pathway (Fig. 4A). This suggests that CtWRKY proteins may mitigate pathogenic infections through interactions with specific MAPK cascades.

The plant exhibits significant variations in total phenolic content and antioxidant capacity among different flower colors, with yellow flowers showing the highest levels [49]. Understanding the regulatory mechanisms of CtWRKY genes that influence phenolic content and antioxidants could enhance the medicinal and economic values of C. tinctorius. In our study of 84 CtWRKY genes in C. tinctorius, distinct upregulation patterns were observed in different flower colors and tissues at various developmental stages (Fig. 6A). This prompts inquiries about the relationship between CtWRKY gene expression and overall phenolic content and antioxidant levels in flowers. This is pertinent to earlier research conducted on other plant species [50, 51].

Responses of CtWRKY genes to exogenous abiotic stress and hormones

In our study, most CtWRKY genes were upregulated in response to cold stress (Fig. 7C). ABA treatment resulted in significant upregulation of only three genes (Fig. 7B), while drought treatment led to significant upregulation of two genes (Fig. 7A). Notably, CtWRKY48 and CtWRKY70 exhibited upregulation in yellow flowers, as well as in LL_DAF10 and LL_DAF20, indicating potential involvement in flavonoid biosynthesis, including anthocyanidin synthase (Fig. 6A; Fig. 6B). Previous research has linked WRKY48 to the regulation of anthocyanin biosynthesis [36]. Additionally, CtWRKY48 and CtWRKY70 may play roles in delaying senescence, similar to WRKY70 in other plants [37, 38], highlighting the need for further investigation into their functions in anthocyanin biosynthesis and response to cold stress. The upregulation of CtWRKY58 and CtWRKY63 in yellow C. tinctorius emphasizes their importance in stress responses, influencing germination, development, and cold stress tolerance (Figs. 6A, B, 7C). Overexpression of CtWRKY58 may affect antioxidant capacity and polyamine content through ABA signaling [49, 51], while CtWRKY63 plays a significant regulatory role during the vernalization-induced flowering process, indicating its involvement in complex regulatory mechanisms [40, 41].

Similarly, CtWRKY7, CtWRKY8 and CtWRKY58 were upregulated in response to cold stress, suggesting their potential roles in the plant's adaptive responses (Fig. 7C). Studies in other species imply their roles in proanthocyanidin synthesis, stress responses, and developmental regulation [42,43,44]. In sesame, WRKY genes are characterized by their remarkable genomic diversity, with over 99% containing multiple introns, extensive multi-domain fusions, and positive selection favoring micro-exons [52]. These features enhance the functional capabilities of WRKY genes, driving key processes such as acquired immunity, ROS signaling, and stress response. Intriguingly, a chloroplast-localized WRKY gene in sesame, Swetha_24868, acts as a master regulator, combining domains such as chorismate mutase and voltage-dependent potassium channels to reduce oxidative stress and bolster immunity. In contrast, our safflower study provides a more targeted focus at WRKY gene expression patterns under various abiotic stresses, identifying specific genes such as CtWRKY44 and CtWRKY72 that are vital for cold and drought tolerance. Moreover, CtWRKY29, which was initially upregulated in LR C. tinctorius, showed downregulation in response to drought stress (Fig. 7B), indicating varied roles in stress responses among plant species. Additionally, CtWRKY15 demonstrated significance in stress responses and the modulation of oleic acid levels (Fig. 6B), potentially alleviating oxidative and endoplasmic reticulum (ER) stresses [45, 46]. Furthermore, CtWRKY72 exhibited upregulation during DAF_0 and DFS, as well as in response to cold stress (Fig. 7C), suggesting its involvement in defense responses and potential roles in flowering and growth regulation [53, 54]. The transient increase in CtWRKY55 expression under cold stress may enhance cold resistance (Fig. 7C), similar to its role in leaf senescence and defense response in Arabidopsis [47]. Additionally, the expression level of CtWRKY65 was peaked during DAF_0 and 7DAG, implying its significance in both seed germination and flower development [55], as well as in the regulation of leaf senescence-associated genes [56].

In summary, our comprehensive findings provide valuable insights into the intricate dynamics of the WRKY gene family in C. tinctorius, highlighting its evolutionary trajectory and functional significance. Understanding gene duplication events and the classification of CtWRKYs into distinct groups enhances our knowledge of the diverse roles and implications of this gene family in safflower molecular processes. These findings are evident in the gene expression profiles observed during responses to abiotic stresses, particularly cold, drought, and ABA treatments.

Conclusions

In conclusion, we identified a total of 84 CtWRKY genes distributed across all 12 chromosomes, and categorized into three main groups based on their phylogenetic relationships. Fragment duplication and tandem duplication exerted an important role in the expansion of the CtWRKY gene family. Analysis of RNA-seq data from four C. tinctorius cultivars, each exhibiting distinct flower colors, revealed complex expression profiles of these genes, suggesting their critical involvement in safflower coloration. Additionally, a protein–protein interaction network analysis conducted for 21 of the 84 CtWRKY genes indicated their interactions with proteins associated with plant stress responses and secondary metabolite biosynthesis. Genes containing LTR, ABRE, and MBS elements demonstrated altered and complex expression patterns in response to cold, ABA, and drought stresses. These findings highlight the diverse roles of CtWRKY genes in various physiological processes essential for safflower growth and development, providing valuable insights into the intricate regulatory networks governing CtWRKY responses to abiotic stresses and developmental processes. The knowledge gained from this study will contribute to advancements in C. tinctorius research and enhance strategies for improving its cultivation under environmental challenges.

Material and methods

Plant materials

C. tinctorius cultivar ‘Yuhonghua 1’ seeds were obtained from the Institute of Chinese Herbal Medicines, Henan Academy of Agricultural Sciences, China. The seeds were selected for uniform size and full maturity before sowing in flowerpots filled with vermiculite. After six days of germination, seedlings were removed from the vermiculite, washed with tap water, and transferred to Hoagland nutrient solution for hydroponic growth. Two weeks later, seedlings with consistent growth were selected for further experiments. For abiotic stress treatments, red flower seedlings were transferred to Hoagland nutrient solution supplemented with 10% PEG6000 and 100 μmol·L−1 NaCl for drought and salt stress, respectively. For low-temperature stress, seedlings were placed in a 4 °C incubator. Leaf samples were collected at 0, 3, 6, 12, and 24 h after treatment, quickly frozen in liquid nitrogen, and stored at −80 °C in an ultralow temperature freezer for RNA extraction. All experiments were conducted in a constant-temperature light incubator set at 25 ± 2 °C with a 16 h-8 h light/dark cycle. Three biological replicates were prepared for each treatment.

Identification of the CtWRKY gene family

The genome sequences of C. tinctorius were obtained from the Genome Database [57]. A BLAST search was conducted using the HMMER version 3.0 hidden Markov model of Pfam [23], with an E-value cutoff of < 1e−5. Additional screening was performed using the Pfam, NCBI conserved domains [24], and SMART database [25] to identify genes with highly conserved amino acids in the WRKY region. Redundant sequences were then eliminated. The WRKY protein sequences of A. thaliana and Oryza sativa were downloaded from Plant Transcription Factor Database (PlantTFDB v5.0, http://planttfdb.gao-lab.org/aboutus.php), while those of H. annuus were download according to a previous study [58]. The WRKY homologs of C. tinctorius were compared with those of A. thaliana, H. annuus, and O. sativa using BLASTP with default parameters. The CtWRKY and AtWRKY, OsWRKY and HaWRKY sequences underwent multiple sequence alignment, phylogenetic and structural analyses.

Phylogenetic analysis and classification of the CtWRKY gene family

The predicted WRKY sequences from A. thaliana were aligned using ClustalX version 2.0 with default parameters. A phylogenetic tree of the CtWRKY sequences was constructed using the neighbor-joining method in MEGA version 6.0 [59]. The parameters for the neighbor-joining method were set as follows: "p-distance" mode, "Complete Deletion" for gap settings, and a bootstrap parameter of 1,000. The subgroups of CtWRKY were determined based on the classification of closely related AtWRKY sequences and the bootstrap support values at relevant nodes.

Protein properties, conserved motifs and gene structures

CtWRKY protein sequences were analyzed using the ExPASy website [26]. The physicochemical properties, such as molecular weights, theoretical isoelectric points (pI), instability index, and subcellular localization, were then assessed. The MEME tools version 5.1.1 was employed to identify up to ten conserved motifs in each CtWRKY protein by using the default parameters [27]. Untranslatted regions (UTR) at the 5’ or 3’ of the gene, coding sequences, and intron positions were determined based on the GFF3 files of A. thaliana and C. tinctorius. Gene structures were visualized using TBtools [8].

Chromosomal mapping and collinearity analysis of CtWRKY genes

The chromosomal positions of CtWRKY genes in C. tinctorius were determined by mapping their gene sequences to the chromosome sequences of C. tinctorius using BLAST programs. The precise gene locations, and tandem duplication were visualized using Mapchart version 2.2 software [60]. Genome data for C. tinctorius, A. thaliana, H. annuus, and O. sativa were obtained from the National Genomics Data Center [61]. Interspecies collinearity analysis between C. tinctorius and A. thaliana, H. annuus, and O. sativa was conducted using MCscanX [62] and TBtools [8]. The final map of C. tinctorius was generated using TBtools [8].

Protein–protein Interaction (PPI) Network

A comprehensive protein interaction network was constructed for all CtWRKY proteins. Homologs of each CtWRKY in C. tinctorius were identified using BLAST, and these homologs were further analyzed by submitting them to the STRING database [63] with default parameters to establish the PPI network. Arabidopsis thaliana was used as the reference species to analyze the interactions of CtWRKY proteins.

Analysis of cis-Regulatory Elements

Promoter sequences for each CtWRKY gene were extracted from the C. tinctorius genome file using SeqKit version 0.13.0 [64]. Briefly, the sequences located upstream to the ATG start codon were retrieved. These promoter sequences were then ana-lyzed for cis-regulatory elements using the PlantCARE website [65].

Expression analysis of CtWRKY genes

In this study, we retrieved RNA-Seq data from the National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA738310. This dataset pertains to four distinct C. tinctorius cultivars characterized by varying flower colors: white (W), yellow (Y), light red (LR), and dark red (DR). Next, transcriptomic analyses of the CtWRKY gene family were performed on the seeds of two cultivars distinguished by their fatty acid profiles: one with high linoleic acid and low oleic acid (HL), and the other with low linoleic acid and high oleic acid (LL). These analyses were conducted at three critical stages of seed development: ovaries at 0 days after flowering (0 DAF), seeds at 10 days after flowering (10 DAF), and seeds at 20 days after flowering (20 DAF). The seed oils from each cultivar were examined at two time points: 10 DAF (HL_DAF10, LL_DAF10) and 20 DAF (HL_DAF20, LL_DAF20). In addition, gene regulation was investigated during seed germination at five stages: cotyledons at 1, 3, 5, 7, and 10 days after germination (1 DAG, 3 DAG, 5 DAG, 7 DAG, and 10 DAG, respectively), using RNA-Seq data from the NCBI BioProject database under accession number PRJNA646045. Finally, gene regulation during flower development was analyzed at five stages: small bud stage (SBS), middle bud stage (MBS), initial flowering stage (IFS), peak flowering stage (PFS), and decayed flowering stage (DFS). In the RNA-Seq experiment, three biological replicates were used. Furthermore, we investigated gene regulation during seed germination at five stages: cotyledons at 1, 3, 5, 7, and 10 days after germination (1 DAG, 3 DAG, 5 DAG, 7 DAG, and 10 DAG, respectively), utilizing RNA-Seq data from the NCBI BioProject database under accession number PRJNA646045. Gene regulation during flower development was analyzed across five stages: small bud stage (SBS), middle bud stage (MBS), initial flowering stage (IFS), peak flowering stage (PFS), and decayed flowering stage (DFS). Each RNA-Seq experiment included three biological replicates.

To assess the expression of the CtWRKY gene family, total RNA was isolated from the samples using TRIzol reagent. The concentration and quality of RNA samples were evaluated using a NanoDrop 2000 spectrophotometer. Subsequently, mRNA was isolated from the total RNA and converted to cDNA using a cDNA synthesis kit. Sequencing adapters were then appended to both ends of the cDNA fragments. The resulting cDNA libraries were sequenced on an Illumina HiSeq 4,000 platform [39]. A quality control step was implemented to eliminate low-quality or erroneous reads from the raw data. The clean reads were aligned to a reference genome using HisAT2, a genome alignment tool, which facilitated the determination of read positional information within the reference genome. This alignment allowed us to explore the expression patterns and quantify CtWRKY genes across various C. tinctorius tissues and cultivars. Gene expression was quantitatively analyzed using fragments per kilobase of transcript per million mapped reads (FPKM), and the FPKM values were then subject to row-scale transformation.

Validation of the expression profiles of CtWRKY genes

The expression levels of CtWRKY genes in response to abiotic stress treatments (ABA, cold, and drought) were assessed using real-time fluorescence-based quantitative PCR (qRT-PCR) assays. The reactions were performed on an FTC-3,000P system (Funglyn Biotech, Toronto, Canada) under the following conditions: initial denaturation at 95 °C for 60 s, followed by 40 cycles of 15 s at 95 °C and 15 s at 60 °C, and a final hold at 4 °C. Gene expression levels were normalized to internal control genes, and relative expression was quantified using the 2−△△Ct method. Each qRT-PCR assay was conducted in triplicate. The primer sequences used in this study are listed in Table S2.

Subcellular localization assay

The full-length coding sequence of CtWRKY60, excluding the stop codon, was cloned and inserted into the pBWA(V)HS-ccdb-GLosgfp vector at Bsa I and Eco31 I restriction sites, under the control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter. The fusion plasmids, along with an empty vector, were transformed into Agrobacterium tumefaciens GV3101. After a three-day infection period, green fluorescence (for GFP) and red fluorescence (for mCherry) were observed using a confocal laser scanning microscope (Nikon C2-ER, Japan). Transformation and infiltration of tobacco were conducted according to a previous study [66]. The primers used for GFP vector construction are listed in Table S2.

Statistical analysis

The correlations between CtWRKY gene family members were assessed through linear regression analysis. All data are presented as mean ± the standard error of the mean. To determine significant differences in expression patterns at different time periods after ABA, cold, and drought treatments, the least significant difference test based on one-way ANOVA analysis was used. All statistical tests were conducted using SPSS software version 21. A p-value of less than 0.05 was considered statistically significant.

Data availability

All data supporting the findings of this study are available within the paper and its supplementary data. The sequences of Carthamus tinctorius L. used in this study were acquired from The Genome Database of Carthamus tinctorius (https://safflower.scuec.edu.cn/).

Abbreviations

C. tinctorius :

Carthamus tinctorius L.

A. thaliana :

Arabidopsis thaliana

O. sativa :

Oryza sativa

C. Melo :

Cucumis Melo L.

C. annuum:

Capsicum annuum

G. hirsutum :

Gossypium hirsutum

TFs:

Transcription factors

UTRs:

untranslated regions

MAPK:

Mitogen-activated protein kinase

MPK4:

Mitogen-activated protein kinase 4

MPK4:

Mitogen-activated protein kinase 3

ACS6:

1-aminocyclopropane-1-carboxylate synthase 6

SIB1:

Sigma factor binding protein 1

NPR1:

Nonexpresser of PR genes 1

SIB2:

Sigma factor binding protein 2

AT1G18a:

Autophagy-related protein 18a

GUN5:

Genomes uncoupled 5

MKS1:

MAP kinase substrate 1

TTG1:

Transparent testa glabra-1

ABA:

Abscisic acid

W:

White

Y,:

Yellow

LR:

Light red

DR:

Deep red

NCBI:

National Center for Biotechnology Information

pI:

Isoelectric points

UTR:

Untranslatted regions

SBS:

Small bud stage

IFS:

Initial flowering stage

PFS:

Peak flowering stage

DFS:

Decayed flowering stage

FPKM:

Fragments per kilobase of transcript per million mapped reads

qRT-PCR:

Quantitative PCR

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Acknowledgements

We appreciate the support of National supercomputing Center in Zhenghou for transcriptome data analysis.

Funding

This research was funded by Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), China Agriculture Research System of MOF and MARA (CARS-21), Major Science and Technology Projects in Henan Province (221100310400), Key Research and Development Program of Henan (241111310200, 231111110800), Henan Academy of Agricultural Sciences Emerging Discipline Development Project (2024XK01); Henan Academy of Agricultural Sciences Independent Innovation Special Fund (2024ZC040); Henan Academy of Agricultural Sciences Outstanding Youth Science and Technology Fund (2024YQ15, 2024YQ16); Henan Center for Outstanding Overseas Scientists (GZS2024025); Henan Province Science and Technology Research Projects (232102110198, 232102110243, 232102110262, 242102110248, 242102110260); Independent Innovation Projects of Henan Academy of Agricultural Sciences (2025ZC45, 2025ZC44).

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Authors and Affiliations

  1. Institute of Chinese Herbel Medicines, Henan Academy of Agricultural Sciences, Zhengzhou , Henan, 450002, China

    Zhengwei Tan, Dandan Lu, Yongliang Yu, Lei Li, Lanjie Xu, Wei Dong, Qing Yang, Chunming Li & Huizhen Liang

  2. Henan Sesame Research Center, Henan Academy of Agricultural Sciences, Zhengzhou , Henan, 450002, China

    Zhengwei Tan, Dandan Lu, Yongliang Yu, Lei Li, Lanjie Xu, Wei Dong, Qing Yang, Chunming Li & Huizhen Liang

  3. State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-Di Herbs, National Resource Center for Chinese Materia Medica, China, Academy of Chinese Medical Sciences , Beijng, 100700, China

    Xiufu Wan

  4. Provincial Key Laboratory of Conservation and Utilization of Traditional Chinese Medicine Resources, Zhengzhou, 450002, Henan, China

    Zhengwei Tan, Lei Li, Lanjie Xu, Wei Dong, Qing Yang, Chunming Li & Huizhen Liang

Authors
  1. Zhengwei Tan
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  2. Dandan Lu
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  3. Yongliang Yu
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  10. Huizhen Liang
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Contributions

ZT and HL conceived the idea, fnished writing this thesis. ZT and DL designed the experiments, ZT and XW completed the bioinformation data analysis. YY, LL, XL WD and YQ performed the experiments, analyzed the data, and completed data visualization. CL performed the subcellular localization assay. All authors read and approved the manuscript.

Corresponding author

Correspondence to Huizhen Liang.

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Ethics approval and consent to participate

The plant material Yuhonghua1 was bred by our research group. All procedures were conducted in accordance with the relevant institutional, national, and international guidelines and legislation. No specific permits were required for plant collection.

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Not applicable.

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The authors declare no competing interests.

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Tan, Z., Lu, D., Yu, Y. et al. Genome-wide identification, characterization and expression analysis of WRKY transcription factors under abiotic stresses in Carthamus tinctorius L. BMC Plant Biol 25, 81 (2025). https://doi.org/10.1186/s12870-025-06079-8

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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