Background

WRKY transcription factors (TFs) modulate plant growth and development and response to abiotic and biotic stress by regulating downstream genes and forming protein complexes [1,2,3,4,5]. WRKY TFs modulate the plant flowering process through various pathways, including photoperiod, autonomous, vernalization, gibberellin, and aging pathways [4, 6]. AtWRKY71 from Arabidopsis thaliana activates FLOWERING LOCUS T (FT) and LEAFY (LFY) to promote flowering [7]. AtWRKY75 interacts with DELLA to activate FT, accelerating flowering [8]. Moreover, WRKY184 from Brassica napus upregulates FRUITFULL (FUL) expression to promote flowering [9]. WRKY TFs are also implicated in phytohormone pathways, such as abscisic acid (ABA) and auxin, to modulate flowering time. OsWRKY72 from Oryza sativa indirectly activates genes associated with the auxin pathway, such as AUXIN1, AUXIN RESISTANT1, and BUD1, and genes related to the ABA pathway, such as ABA2 and ABA INSENSITIVE 4 (ABI4), to promote flowering [10]. WRKY TFs regulate salt and cadmium stress to modulate flowering time. For instance, RtWRKY23 from Reaumuria trigyna upregulates HISTONE ACETYLTRANSFERASE 1, ANT, and MADS AFFECTING FLOWERING 5 expression to alleviate salt stress, resulting in earlier flowering [11]. AtWRKY12 and AtWRKY13 potentially co-regulate flowering time and cadmium stress due to their similar regulatory patterns in modulating flowering time and cadmium stress [4].

Environmental changes, such as nutrient availability, drought stress, and temperature stress, can alter flowering time [4]. However, the role of WRKY TFs in regulating these pathways ultimately modulating flowering time is unclear. This indicates that WRKY TFs may regulate flowering time through additional flowering pathways. Therefore, it is imperative to conduct research to identify flowering-time genes at the genome level and verify whether WRKY TFs regulate these genes. A plant flowering-time gene database, PFGD, was established in the recent past [12]. The database provides a platform to identify WRKY TFs that regulate downstream genes and the proteins associated with these TFs.

Plants in the Arachis genus serve as oil, forage and turfgrass crops [13]. Cultivated peanut (Arachis hypogaea) and Arachis monticola are allotetraploids obtained by crossing by two wild diploids, Arachis duranensis and Arachis ipaensis [14, 15]. Comparative genomic analyses between diploid and tetraploid peanuts reveal asymmetric evolution in the subgenomes of cultivated peanuts [16,17,18]. Asymmetric evolution of subgenomes leads to functional bias of genes in a specific subgenome [16, 17, 19]. A homoeologous WRKY pair, AhTWRKY24 from subgenome B and AhtWRKY106 from subgenome A, were identified in Arachis hypogaea cv. Tifrunner [20]. DNA affinity purification sequencing data revealed that AhTWRKY24 and AhtWRKY106 regulate approximately an equal number of downstream genes in A. hypogaea cv. Tifrunner genome, but they also exhibit specific regulation of distinct downstream genes [20]. These results indicate that asymmetric evolution influences genes regulated by WRKY in A. hypogaea cv. Tifrunner.

Although WRKY TFs have been identified at the genome level in members of Arachis genus [5, 21], their role in regulating flowering time is yet to be fully elucidated. In this study, flowering-time genes for A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner were retrieved from PFGD database. This study revealed the regulatory networks of WRKY TFs in the four Arachis species through bioinformatics approaches.

Methods

Sequence retrieval

The PFGD database (http://pfgd.bio2db.com/index.html) is a valuable repository for genes that regulate flowering time in plants [12]. The flowering-time genes of the Arachis species were retrieved from the PFGD database. The species of the Arachis genus included A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner.

WRKY TFs have been identified in various Arachis species [5, 21]. A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner have 16 WRKY TFs, which are AtWRKY12 and AtWRKY75 homologs and are involved in regulating flowering time [5, 21, 22]. The 16 WRKY TFs in the four Arachis species were retrieved from PeanutBase database based on previous studies [5, 21, 22].

The orthologs of the genes in A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner were identified using the MCScan X program with an e-value of 1E-10 [23]. Similarly, paralogs and homoeologs were identified using the MCScan X program. The homoeologs were identified in A. monticola and A. hypogaea cv. Tifrunner.

Identification of WRKY TFs regulation flowering-time genes in Arachis genus

The 2 kb cis-acting regions of flowering-time genes were isolated from the four Arachis species and uploaded to the Nesite database to predict the WRKY TF binding sites (TFBSs) [24]. The search parameters were an expected mean number of 0.01, a statistical significance level of 0.95, an 80% homology between known TFBS and motif, and a 20% variation in the distance between TFBS blocks. The protein interaction relationships between the 16 WRKY TFs and flowering-time proteins in the four Arachis species were predicted using the STRING database. A. thaliana was used as a reference and the protein–protein interaction network analysis was conducted with the default parameters in the STRING database.

Tissue expression profile of WRKY and flowering-time genes in A. hypogaea cv. Tifrunner

The RNA-seq datasets of 22 tissues of A. hypogaea cv. Tifrunner were retrieved from PeanutBase [25, 26]. The raw read counts were aligned to the A. hypogaea cv. Tifrunner genome using Bowtie 2 in the TBtools program [23], and expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) using RSEM [27]. The expression levels were standardized by log2 transformation (FPKM + 1). The expression patterns were visualized using TBtools program [23].

Phylogenetic analysis

MAFFT was used to conduct multiple sequence alignments with default parameters [28]. ProtTest was used to estimate the best-fit model for the construction of a phylogenetic tree based on the maximum likelihood method [29]. The IQ-tree program was used to construct the maximum likelihood trees using the best-fit model from the ProtTest program, 10,000 ultrafast bootstraps, and 1000 the SH-like approximate likelihood ratio test. The phylogenetic trees were visualized using the Figtree tool [30].

Results

Multiple flowering-time pathways are identified in Arachis genus

The PFGD database comprises 552, 622, 514, and 576 flowering-time genes from A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner (Accessed on January 23, 2024). Notably, analysis of the cis-acting elements showed that 41, 53, 81, and 91 flowering-time genes are potentially regulated by WRKY TFs in A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner (Fig. 1 and Additional File 1). These four species of Arachis genus had similar flowering pathways regulated by WRKY TFs. The flowering pathways included aging, autonomous, circadian clock, hormone, photoperiod, sugar, temperature, and vernalization. However, no  previous evidence confirms the involvement of WRKY TFs in sugar and temperature pathways to modulate flowering time.

Fig. 1
figure 1

The flowering-time genes regulated by WRKY transcription factors in the four Arachis species

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Homologous flowering-time genes have asymmetric WRKY transcription factor binding sites

The four species in Arachis genus have different numbers of flowering-time paralogs. In A. duranensis, four pairs of paralogous flowering-time genes contained WRKYTFBSs, whereas 15 flowering-time genes from 15 paralogous pairs contained WRKY TFBSs (Fig. 2 and Additional File 2). Similarly, A. ipaensis and A. hypogaea cv. Tifrunner had two and six pairs of paralogous flowering-time genes containing WRKY TFBSs, respectively (Fig. 2 and Additional File 2). Moreover, one copy of 12, 13, and 19 paralogous flowering-time gene pairs in A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner exhibited WRKY TFBSs (Fig. 2 and Additional File 2). These results indicate that evolutionary patterns of WRKY TFBSs in flowering-time genes vary across species.

Fig. 2
figure 2

Homologs potentially regulated by WRKY transcription factors. a A schematic representation of symmetric and asymmetric WRKY transcription factor binding site (TFBS) in a homologous pair. b The homologs of flowering-time genes exhibiting WRKY TFBSs in the four Arachis species

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The findings showed that A. monticola and A. hypogaea cv. Tifrunner have varying number of homoeologs associated with flowering time. A. monticola and A. hypogaea cv. Tifrunner exhibited two and 14 homoeologous flowering-time gene pairs containing WRKY TFBSs, respectively. Conversely, A. monticola and A. hypogaea cv. Tifrunner had one copy of 15 and 74 homoeologous flowering-time gene pairs exhibiting WRKY TFBSs (Fig. 2 and Additional File 2). These results indicate the WRKY TFBSs in flowering-time genes exhibit an asymmetric evolutionary pattern between the two species.

Previous studies demonstrated that 16 Arachis WRKY TFs are orthologs with AtWRKY12 and AtWRKY75, which regulate flowering time by binding W-box elements of FUL and FT genes [8, 31]. Conserved orthologs of flowering time were identified across four Arachis species through synteny analyses. These genes are mainly implicated in aging, autonomous, and sugar pathways (Table 1). These results indicate that WRKY TFs modulate specific regulatory networks of flowering time in the four Arachis species.

Table 1 WRKY transcription factors potentially regulate conserved orthologs in the four Arachis species
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WRKY TFs interact with FUL and AP2 to modulate flowering time in Arachis genus

The WRKY TF interaction relationships among the four Arachis species were predicted using the STRING database. The WRKY TFs, which were orthologs to AtWRKY12, interacted with FUL in the four Arachis species (Fig. 3). Similarly, the WRKY TFs, which were orthologs to AtWRKY75, interacted with APETALA2 (AP2) in the four Arachis species (Fig. 3). Phylogenetic analyses showed that WRKY TFs and the associated proteins exhibited different evolutionary relationships in the four Arachis species. FUL showed a high likelihood of loss in A. hypogaea cv. Tifrunner genome compared to the WRKY12 TFs (Fig. 3). Conversely, AP2 was prone to expansion relative to WRKY75 in A. monticola and A. hypogaea cv. Tifrunner genomes (Fig. 3). Notably, there was no evidence confirming that AtWRKY12 and AtWRKY75 proteins interact with FUL and AP2 to modulate flowering time. The results indicate that WRKY TFs exhibit specific protein interaction relationships to modulate flowering time in the four Arachis species.

Fig. 3
figure 3

WRKY transcription factors interact with FUL and AP2 to modulate flowering time in the four Arachis species

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Varying expression patterns of flowering-time genes in 22 tissues of A. hypogaea cv. Tifrunner

WRKY TFs regulating flowering time clustered in group I based on their expression levels in 22 tissues of A. hypogaea cv. Tifrunner (Fig. 4). The genes in group I exhibited low expression levels compared to other groups (Fig. 4). Group I had two homoeologous gene pairs, AhTWRKY35 and AhTWRKY116, and AhTWRKY69 and AhTWRKY149. Each homoeologous gene pair exhibited similar expression pattern in 22 tissues (Fig. 4). These results indicate that WRKY homoeologous gene pair share similar regulatory networks. In addition, 18 flowering-time genes were grouped in a subclade based two the WRKY homoeologous gene pairs (Fig. 4), indicating that these genes are potentially regulated by these WRKY homoeologs.

Fig. 4
figure 4

The expression patterns of WRKY and flowering-time genes in 22 tissues of Arachis hypogaea cv. Tifrunner. Blue font indicates interacting proteins with WRKY transcription factors

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In addition, AhTWRKY49 and AhTWRKY99 (ortholog with WRKY75) indicated potential synergistic interactions with 38RLY4 (AP2), and AhTWRKY35 and AhTWRKY116, and AhTWRKY69 and AhTWRKY149 (ortholog with WRKY12) exhibited potential synergistic interactions with WIWA1B and IQR3N2 (FUL), because they had similar expression patterns (Figs. 3 and 4). Conversely, AhTWRKY49 and AhTWRKY99 exhibited potential antagonistic interactions with 00XS3D, 095TGF, and H1PWIN (AP2) due to the differences in expression patterns (Figs. 3 and 4).

Discussion

Arachis plants are essential sources of oil, proteins, and forage [13]. The genomes of cultivated peanut and its progenitors have been sequenced [15, 17, 32,33,34]. WRKY TFs have been identified at the genome level in members of Arachis genus [5, 21]. However, the functions of WRKY TFs in flowering time have not been elucidated. In this study, WRKY TFs that regulate flowering-time genes and their interaction proteins in A. duranensis, A. ipaensis, A. monticola, and A. hypogaea cv. Tifrunner were identified using bioinformatics approaches. The main findings are summarized as follows: Firstly, WRKY TFs are involved in aging, autonomous, circadian clock, hormone, photoperiod, sugar, temperature, and vernalization pathways to modulate flowering time in the four Arachis species. Secondly, asymmetric WRKY TFBSs were identified in homologs of the flowering-time genes across the four Arachis species. Thirdly, two conserved protein complexes involving WRKY TFs interaction with FUL and AP2 modulated flowering time in the four Arachis species.

WRKY TFs are primarily involved in flowering pathways and phytohormone pathways to modulate flowering time [4, 35]. The flowering pathways include photoperiod, autonomous, vernalization, gibberellin, and aging pathways [4, 6]. WRKY TFs regulate flowering time through ABA, auxin, and ET pathways [4, 10, 36]. However, the role and the underlying mechanisms of WRKY TFs in regulating flowering time through sugar and temperature pathways have not been fully elucidated. Previous studies demonstrated that low sugar and high temperature promote flowering, whereas high sugar and low temperature conditions delay flowering in plants [4, 37,38,39]. These findings imply that WRKY TFs potentially modulate flowering time through sugar and temperature pathways in the four Arachis species.

Specific interactions between WRKY TFs and other proteins modulated flowering time in the four Arachis species. Arachis WRKY TFs interacted with FUL and AP2 proteins to modulate flowering time. FUL, a member of the MADS-box gene family, modulates flowering time and floral meristem development [40, 41]. AP2, a member of the AP2/ERF gene family, regulates the interaction between floral meristem and APETALA1 (AP1), LEAFY (LFY), and CAULIFLOWER (CAL) to modulate flower and floral meristem development [42, 43]. Notably, a regulatory network exists between FUL and AP2. FUL directly and negatively regulates AP2 to downregulate wuschel-related homeobox expression in shoot apical meristem, reducing flowering in monocarpic plants [44]. Furthermore, AP2 downregulates genes implicated in axillary bud dormancy and cytokinin signaling, resulting in global proliferative arrest to affect flowering in monocarpic plants [45]. These findings indicate that WRKY TFs are implicated in the global proliferative arrest pathway in Arachis genus.

Arachis WRKY TFs, which regulate flowering time, are orthologs with AtWRKY12 and AtWRKY75 [5]. AtWRKY12 and AtWRKY75 interact with DELLA proteins to modulate flowering time [8, 31]. Moreover, AtWRKY12 activates FUL, and AtWRKY75 activates FT to promote flowering [8, 31]. Based on these results, we hypothesized that Arachis WRKY TFs, orthologous to AtWRKY12, interact with FUL protein and regulate FUL expression.

However, our study had some limitations. Firstly, it is challenging to establish one-to-one regulatory relationships between WRKY TF and downstream genes using bioinformatics approaches. This is because WRKY TFs regulate a conserved W-box element shared across several downstream genes. Additionally, our results showed that the number of downstream genes was higher than the number of WRKY TFs in the four Arachis species. These findings indicate that one WRKY TF may regulate several downstream genes to modulate flowering time. AtWRKY63 activates COOLAIR and COLDAIR, leading to the downregulation of FLOWERING LOCUS C (FLC) expression and consequently accelerating flowering [46]. AtWRKY71 activates FT and LFY to promote flowering [7]. Our results demonstrated that WRKY TFBSs of homologous downstream genes exhibit asymmetric evolution, suggesting that WRKY TFs interact with other transcription factors to modulate the flowering process in the four Arachis species.

Conclusion

In summary, bioinformatics approaches were used in this study to predict the WRKY TFs regulating downstream genes and interaction proteins in the four Arachis species. WRKY TFs are involved in multiple pathways to modulate flowering time in the four Arachis species. WRKY TFs can interact with FUL and AP2 modulated flowering time in the four Arachis species. Although several novel regulatory networks were elucidated in this study, further experimental testing is required to verify these relationships.