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Genome-wide identification and expression analysis of the WRKY gene family reveal essential roles in abiotic stress responses and polysaccharides and flavonoids biosynthesis in Platostoma palustre (Blume) A. J. Paton

BMC Plant Biology volume 24, Article number: 1122 (2024) Cite this article

Abstract

Background

Platostoma palustre (Blume) A. J. Paton is an economic crop with medicinal and edible properties. WRKY transcription factors are widely involved in the growth and development, response to adversity stress, and hormone transduction of plants. The identification of the WRKY gene family has been completed in many plants, however, it has not yet been identified and analyzed at the genome-wide level in P. palustre.

Results

In this study, we identified 133 PpWRKY gene family members (PpWRKYs) at the whole genome level of P. palustre, which were unevenly distributed on 15 chromosomes. Based on their protein structure and phylogenetic characteristics, the 133 PpWRKYs were divided into 3 subgroups. Segmental duplication events might play a crucial role in the expansion of the PpWRKY gene family. Through the transcriptome expression data analysis, the expression profiles of PpWRKY genes under Cd, red light, salt, and drought stresses were analyzed in this study, suggesting that WRKY transcription factors may play a crucial role in responding to different abiotic stresses in P. palustre. Notably, PpWRKY92 exhibited simultaneous responses to Cd, light intensity, salt, and drought stresses. Additionally, PpWRKY21, 75, 90, 52, 124, 39, 115, 122, 20, and 76 demonstrated a strong correlation with both monosaccharides and flavonoids. Taken together, PpWRKY20, 39, 75, 76, 90, 92, 115, 122, and 124 were found to be associated with the abiotic stress response and polysaccharides and flavonoids biosynthesis in P. palustre, except the low-expressed PpWRKY21 and 52.

Conclusion

The present study laid the foundation for the abiotic stress response and metabolite regulation of this gene family in P. palustre.

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Background

Platostoma palustre (Blume) A. J. Paton, also known as Mesona chinensis Benth, is an annual plant belonging to the Platostoma genus within the Lamiaceae family. It is an economic crop with medicinal and edible properties, found widely in the tropics and subtropics, including the Southeast Asian countries and South China [1, 2]. In China, it is widely cultivated in Guangxi, Guangdong, Jiangxi, Zhejiang, Fujian, and Taiwan provinces [3]. P. palustre contains a variety of bioactive compounds, including polysaccharides, flavonoids, volatile oils, phenolic acids, etc [4]. Which exhibit antioxidant [5], antiviral [6], hypoglycemic [7], and lipid-lowering [8] properties. P. palustre is often utilized as an ingredient in refreshing summer delicacies and herbal tea beverages and serves as a raw material for various products such as lipid-lowering teas, hypoglycemic agents, novel coagulants, and purely natural food colorants [9].

The WRKY gene family is unique to plants, and its members are named after the conserved “WRKYGQK” sequence, which plays an important regulatory role in the response to plant stress [10, 11]. Based on the differences in the number of WRKY structural domains and zinc finger motifs, the WRKY gene family can be divided into three major groups, Group I to II. Among them, Group II can be further classified into five subgroups (a, b, c, d, and e) depending on the differing numbers of amino acids [12]. Currently, an increasing number of WRKY gene family members are being identified from various plants. In Rheum palmatum, 53 WRKY genes have been identified, with 7 genes (RpWRKY5/12/25/28/30/42/47) exhibiting high expression levels in both roots and rhizomes, potentially implicated in their growth and development processes [13]. In Aquilegia viridiflora, 35 WRKY genes have been discovered, with AvWRKY3 and AvWRKY7 playing pivotal roles in the plant’s salt stress response [14]. In the rice genome, a total of 103 genes encoding WRKY transcription factors have been identified, with 19 of these genes exhibiting altered expression under drought stress [15]. In Yulania biondii, 56 WRKY genes have been identified, including MBI19961_MAGBIO and MBI047_MAGBIO, which are highly expressed in roots and flowers, respectively. As roots and flowers are the primary tissues for drug synthesis in this species, it is hypothesized that these two genes may regulate secondary metabolism in these tissues [16].

The WRKY transcription factor family genes have been identified across various plant genomes, including Arabidopsis thaliana [17], soybean [18], Baphicacanthus cusia [19], Coptis chinensis Franch [20], Rheum palmatum [13], etc. However, research on the WRKY transcription factor family in P. palustre remains scarce, and the underlying mechanisms of its function are yet to be elucidated. This study identified and analyzed the members of the WRKY gene family using bioinformatics methods. Furthermore, we investigated the expression patterns of these genes across different tissues at various developmental stages, as well as under salt, drought, light intensity, and Cd stress treatments. The present study laid the foundation for the abiotic stress response and metabolite regulation of this gene family in P. palustre.

Materials and methods

Identification of the WRKY genes family

The WRKY family protein sequences of Arabidopsis thaliana and Scutellaria baicalensis were used as reference sequences. The HMMER 3.0 software was employed to construct the hidden Markov model for the acquired P. palustre WRKY family sequences. All coding protein sequences were searched with this model and all potential WRKY family sequences were found in their protein sequences. Additionally, the blastp (version: ncbi-blast-2.10.1+) [21] was utilized to align all the protein sequences of P. palustre with the WRKY family sequences of A. thaliana and S. baicalensis (e-value was set to 1e− 5). Sequences that aligned successfully were deemed potential WRKY family members. Subsequently, the identified sequences were combined to form a pool of candidate WRKY family protein sequences. Domain annotation of the candidate sequences was performed using the pfamscan (version: v1.6) software and Pfam A (version: v33.1) [22, 23] database. Sequences containing the PF03106 domain were identified as the final WRKY sequences.

Analysis of conserved motifs, chromosomal location information, and physicochemical properties

The conserved motifs of the P. palustre WRKY family genes were analyzed using the online website MEME (http://meme-suite.org/, v5.0.5) (Parameters: -protein, -nmotifs: 15, -o: rea1-minw: 6, -maxw: 50, -mod: zoops). Using the GFF3 annotation file of the P. palustre genome, the positional information for introns, exons, and chromosomal locations of PpWRKYs was extracted. This information was then used to generate gene structure and chromosomal localization maps. Additionally, the online tool ExPASy (http://web.expasy.org/protparam) was utilized to generate both the gene structure and chromosome positioning maps. The physicochemical properties of the PpWRKY family members were analyzed using the online website ExPASy.

Phylogenetic analysis

Multiple sequence alignments of WRKY protein sequences from P. palustre, A. thaliana, and S. baicalensis were conducted using MAFFT v7.427 with default parameters. The Neighbor-Joining (NJ) phylogenetic tree was constructed using MEGA 10.0 software [24], with a bootstrap value of 1,000, and visualized utilizing FastTree (v2.1.3).

Cis-acting elements analysis

The upstream 2000 bp sequence of the PpWRKY genes was extracted, and potential cis-acting elements were predicted utilizing the online tool PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Collinearity analysis

Utilizing the P. palustre genome and chromosomal position data of PpWRKYs, MCScan X was employed to perform collinearity analysis on PpWRKYs, and the results were visualized using Tbtools (v1.1045) software.

Prediction of protein interaction networks

An online tool, String (https://cn.string-db.org/), was utilized to construct a protein interaction network model for the PpWRKY family, setting the species parameter to A. thaliana and specifying a species confidence level of 0.40.

Expression profiling

The expression profiling of the WRKY gene family was analyzed utilizing the Majorbio Cloud Platform (https://cloud.majorbio.com/page/tools/) with the transcriptome data previously generated by our team. The transcriptome data encompassed various tissues of P. palustre at distinct developmental stages, specifically leaves (LS1, LS2, LS3) and stems (SS1, SS2, SS3) (Table S1). Furthermore, expression profiling encompassed diverse Cd concentrations (0, 25, 50, 100 µmol/L) and red light intensities (T2: two LED tubes; T4: four LED tubes; T6: six LED tubes; T8: eight LED tubes). Lastly, the expression profiling was also analyzed under salt (0, 150mM NaCl) and drought (0, 15% PEG6000) stress conditions. The fastp software was used to perform quality control on raw RNA-seq data. The specific steps and sequence were as follows: (1) Remove adapter sequences from the reads and discard reads without inserted fragments due to adapter self-ligation or other reasons. (2) Trim low-quality bases from the beginning (5’ end) of the sequences (average quality value less than 20, calculated using a sliding window size of 4) and from the end (3’ end) of the sequences (quality value less than 3). (3) Remove reads containing N (ambiguous bases): reads with more than 5 N are discarded. (4) Discard sequences that are shorter than 30 bp after adapter removal and quality trimming. A gene’s fold change (FC) value greater than 2 (FC > 2) indicated upregulation, while an FC value less than 1/2 (FC < 1/2) indicated downregulation.

Correlation analysis

The correlation cluster analysis was conducted using the Metware Cloud Platform (https://cloud.metware.cn) between the transcriptional expression data of WRKY family genes and the monosaccharide and flavonoid metabolome data [25] collected from various tissues at different developmental stages of P. palustre, using the Pearson method with a p-value threshold of < 0.05 and a correlation coefficient threshold of 0.90.

Three-dimensional protein structure prediction of PpWRKYs

The protein sequences were input into SWISS-MODEL (https://swissmodel.expasy.org/) for homology modeling to predict the tertiary structures of PpWRKYs.

Expression of the PpWRKY92 gene under salt and drought stress

Normal-growing and uniform-sized seedlings of P. palustre were selected and hydroponically grown in 150 mM NaCl and 15% PEG6000 for salt and drought treatments, respectively. Roots, stems, and leaves of the stress-treated 0, 6, 12, and 24 h plants were harvested for RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) analysis, respectively. For qRT-PCR analysis, total RNA was extracted from each sample using the FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). The primers used for qRT-PCR analysis are listed in Table S2. The qRT-PCR program was as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. Each reaction contained three technical and three biological replicates. Relative gene expression levels were analyzed by Excel software using the 2−ΔΔCt method.

Statistical analysis

SPSS 23.0 software was utilized for statistical analysis, and the mean values were assessed using Duncan’s multiple range test to determine statistical significance (p < 0.05).

Results

Identification and physicochemical properties of the PpWRKY gene family

In this study, a total of 133 WRKY genes were identified in the genome of P. palustre, which were sequentially named PpWRKY1-133 based on their gene IDs (Table 1). The coding sequences (CDs) of PpWRKY gene family members ranged from 318 to 2244 bp in length, with amino acid counts varying from 105 to 747 aa. Their relative molecular weights spanned 11681.21 to 80391.53 Da, isoelectric points (PIs) ranged from 4.86 to 9.92, and GRAVY values were less than 0. Apart from PpWRKY31, PpWRKY32, PpWRKY37, PpWRKY60, PpWRKY67, PpWRKY76, PpWRKY110, and PpWRKY132, which were identified as stable proteins, the remaining PpWRKY proteins were classified as unstable hydrophilic proteins, characterized by instability coefficients exceeding 40.

Table 1 Physicochemical properties of WRKYs in Platostoma palustre
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Chromosomal localization and collinearity analysis of the PpWRKY gene family

The chromosomal localization analysis revealed an uneven distribution of 133 PpWRKY genes across the 15 chromosomes, with PpWRKY133 not assigned to any specific chromosome (Fig. 1). The maximum number of PpWRKY genes (19) was observed on chromosome 9, whereas the minimum number (1) was found on chromosome 10. The PpWRKY genes were distributed randomly on the P. palustre chromosome. Furthermore, to gain insights into the evolutionary mechanisms and expansion patterns of the PpWRKY gene family, we conducted a collinearity analysis. Our results indicated the occurrence of 112 segmental duplication events within the PpWRKY gene family (Fig. 2), including a notable example between PpWRKY101 on chromosome 1 and PpWRKY44 on chromosome 11. Additionally, we identified 4 tandem duplication events within the PpWRKY gene family, occurring specifically on chromosomes 2, 6, 9, and 15. These findings implied that the evolution of the PpWRKY gene family may be primarily driven by segmental duplications.

Fig. 1
figure 1

Chromosomal distribution of the PpWRKY gene family

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Fig. 2
figure 2

Synteny analysis of PpWRKY genes. Gray lines indicate all synteny blocks in the P. palustre genome, while the colored lines indicate the duplication of PpWRKY gene pairs

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Phylogenetic relationships of PpWRKY gene family

To elucidate the evolutionary relationship among PpWRKYs, AtWRKYs, and SbWRKYs, a phylogenetic tree was constructed using the MEGA10 software. The phylogenetic tree constructed in this study included 133 PpWRKYs, 72 AtWRKYs, and 77 SbWRKYs (Fig. 3). A total of 282 WRKY proteins were categorized into three distinct groups: I, II, and III. Notably, Group II was further subdivided into six subgroups: IIa, IIb, IIc, IId, IIe, and IIf. The PpWRKYs were evenly distributed across these groups. Group I comprised 51 WRKY members, with 23 PpWRKYs, 14 AtWRKYs, and 14 SbWRKYs. Conversely, Group III contained 14 PpWRKYs, 13 AtWRKYs, and 9 SbWRKYs. Group II encompassed 195 WRKY members, with IIf having the smallest number of members (12, including 3 PpWRKYs, 2 AtWRKYs, and 7 SbWRKYs). Conversely, IIc had the largest number (70, with 38 PpWRKYs, 16 AtWRKYs, and 16 SbWRKYs). Additionally, IIa, IIb, IId, and IIe contained 16, 36, 26, and 35 WRKY members, respectively.

Fig. 3
figure 3

Phylogenetic tree of WRKYs from P. palustre, S. baicalensis, and A. thaliana. Different colors represent different groups. The stars represent PpWRKY genes, triangles represent AtWRKY genes, and circles represent SbWRKY genes

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Analysis of conserved motifs and gene structure

Our analysis of conserved motifs in PpWRKYs identified 15 motifs among 133 sequences (Fig. 4), further revealing the diversity of PpWRKY genes. Except PpWRKY31 and PpWRKY35 lacking motif1, and PpWRKY19, PpWRKY46, PpWRKY58, and PpWRKY59 devoid of motif2, all 127 PpWRKYs contained motifs 1 and 2, presumed to be the core conserved motifs of PpWRKY proteins. Specifically, motifs 4, 8, 10, 11, 12, and 13 were found in 15, 14, 7, 8, 11, and 8 PpWRKY proteins, respectively. Notably, motif2 was exclusively present in PpWRKY31 and PpWRKY35, albeit with varying distribution positions.

Fig. 4
figure 4

Phylogenetic tree, gene structures, and protein motifs of PpWRKYs

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Gene structure analysis results showed that the number of exons in group I genes ranged from 3 to 7, while those in groups IIa, IIb, and IIc ranged from 3 to 5, 4 to 9, and 2 to 4, respectively. Members of groups IId, IIf, and III, as well as most of group IIe, had three exons. Genes within the same group exhibited structural similarities, although there were notable variations in the intron and exon counts across different groups, indicating a higher complexity in the structure of PpWRKY genes.

Analysis of promoter cis-acting elements of PpWRKY family genes

The analysis of promoter cis-acting elements was conducted to investigate the potential biological functions of PpWRKY genes. As shown in Fig. 5, multiple cis-acting elements including light-responsive elements, hormone-responsive elements, stress-responsive elements, and development-responsive elements were identified in the promoter regions of the majority of PpWRKY genes. Among these, a total of 1577 light-responsive elements were found throughout the promoter regions of all PpWRKY genes. In addition, the promoter regions of PpWRKY genes contained 1124 hormone-responsive elements, 513 stress-responsive elements, and 235 development-responsive elements (Table S3). The hormone-responsive elements included elements like methyl jasmonate-responsive, abscisic acid-responsive, and gibberellin-responsive elements. The stress-responsive elements contained elements related to low-temperature responsive regulation, enhancer-like elements involved in anoxic specific inducibility, and so on. The development-responsive elements encompassed a regulatory element related to maize alcohol-soluble protein metabolism, one for meristematic tissue expression, a circadian rhythm regulatory element, and so on. These results suggested that WRKY genes might have important roles in the growth, development, and response to abiotic stress in P. palustre.

Fig. 5
figure 5

Cis-acting element analysis of PpWRKY promoter regions

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Prediction of protein interaction networks of PpWRKY genes

The results indicated that 37 proteins participated in the interactions, forming a total of 139 interaction groups (Fig. 6). It was observed that PpWRKY35, PpWRKY39, PpWRKY58, PpWRKY122, and PpWRKY132 were not part of these interactions, suggesting that these proteins may function autonomously in regulatory roles.

Fig. 6
figure 6

Protein interaction network analysis of PpWRKYs. Inside the nodes, the protein 3D structures are visible, and protein-protein interactions are represented by lines. Confidence score > 0.4

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Spatiotemporal expression patterns of PpWRKY genes

Based on transcriptome data, we analyzed the expression patterns of PpWRKY gene family members in stems and leaves across various growth stages. Our results showed that 133 PpWRKY genes were generally under-expressed in P. palustre stems and leaves (Fig. 7A). Four PpWRKY genes (PpWRKY17, PpWRKY31, PpWRKY59, and PpWRKY100) were completely absent from expression in stems and leaves, while 34 PpWRKY genes showed minimal expression. High expression levels of PpWRKY5, PpWRKY53, and PpWRKY121 were observed in SS3 and LS1-LS3. In particular, PpWRKY81 exhibited the highest expression levels in SS1-SS3 and LS1-LS3.

Fig. 7
figure 7

Correlation and cluster analysis between the expression of PpWRKYs and the content of monosaccharides and flavonoids. Heat map of the expression patterns of PpWRKY genes (Log2(FPKM + 1)) across different tissues and growth stages (A). Correlation and cluster analysis between the expression of PpWRKYs and the content of monosaccharides (B). Correlation and cluster analysis between the expression of PpWRKYs and the content of flavonoids (C)

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Correlation and cluster analysis between the expression of PpWRKYs and the content of monosaccharides and flavonoids

Studies have reported that P. palustre polysaccharides contained D-galactose, D-galacturonic acid, D-glucuronic acid, D-glucose, D-mannose, D-ribose, D-arabinose, L-xylose, etc [25,26,27,28]. P. palustre flavonoids included quercetin, isoquercetin, rutin, and astragalin [29,30,31,32,33]. To further elucidate the potential relationship between PpWRKYs and the quality and medicinal effects of P. palustre, we conducted correlation and cluster analyses between the expression of 133 PpWRKYs and the content of monosaccharides and flavonoids (Fig. 7B, C). Correlation analysis revealed that 13 PpWRKY genes (PpWRKY20, 21, 39, 50, 51, 52, 65, 75, 76, 90, 115, 122, 124) were significantly associated with monosaccharides in P. palustre (Fig. 7B). Furthermore, analysis uncovered that 13 PpWRKY genes (PpWRKY16, 20, 21, 39, 52, 71, 75, 76, 90, 115, 120, 122, 124) showed a correlation with flavonoids in P. palustre (Fig. 7C). In particular, PpWRKY20, 21, 39, 52, 75, 76, 90, 115, 122, and 124 demonstrated a strong correlation with both monosaccharides and flavonoids. Of these, PpWRKY21 and 52 exhibited a low expression level. Therefore, we hypothesized that the remaining eight PpWRKYs might play a role in regulating the biosynthesis of polysaccharides and flavonoids in P. palustre.

Expression patterns of PpWRKYs under abiotic stresses

To further clarify whether the PpWRKY gene family responds to different abiotic stresses, we analyzed the expression profiles of PpWRKY gene family members under Cd, light, salt, and drought stresses using the RNA-Seq data of P. palustre (Fig. 8A, B, C, and D). The results showed that a total of 125 PpWRKYs were responsive to Cd stress and different gene members showed different expression patterns with increasing Cd concentration. Specifically, the expression of 9, 16, and 18 PpWRKYs was significantly up-regulated, while 19, 22, and 21 PpWRKYs were significantly down-regulated at Cd25, Cd50, and Cd100 concentrations, respectively (Fig. 8A). Except for PpWRKY23, PpWRKY31, PpWRKY59, PpWRKY70, PpWRKY98, PpWRKY100, PpWRKY110, and PpWRKY133, which were not expressed at all, the remaining 125 PpWRKYs showed varied expression patterns with increasing light intensity (Fig. 8B). Under salt and drought stresses, 32 and 8 PpWRKYs were up-regulated in gene expression, while 17 and 18 PpWRKYs were significantly down-regulated, respectively (Fig. 8C, D). Notably, PpWRKY92 could respond to Cd, light, salt, and drought stresses simultaneously, suggesting its pivotal role in the response of different abiotic stresses in P. palustre.

Fig. 8
figure 8

Heat map of the expression patterns of PpWRKY genes under different abiotic stresses, including Cd (A), Light intensity (B), NaCl (C), and PEG6000 (D). Expression levels (Log2(FPKM + 1)) are indicated by the color scale

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Spatio-temporal expression pattern of the PpWRKY92 gene under salt and drought stresses in P. Palustre

The expression of PpWRKY92 under salt and drought stresses was further detected by qRT-PCR (Fig. 9). The results indicated that the expression of PpWRKY92 exhibited significant organ-specific differences under salt and drought stresses. Under salt stress, the expression of the PpWRKY92 gene was significantly upregulated in the stem, leaves, and roots (except for NaCl-stem, 6 h). Under drought stress, the expression of the PpWRKY92 was significantly downregulated in the roots, while in the leaves and stems at 6 h of treatment, the expression of PpWRKY92 was significantly upregulated, with the highest expression levels observed.

Fig. 9
figure 9

qRT-PCR expression analysis of PpWRKY92 gene under salt (A, B, C) and drought (D, E, F) stress conditions. Error bars represent the standard error of three biological replicates and asterisks indicate a significant difference between the treatment and CK group (*p < 0.05, ** p < 0.01, *** p < 0.001)

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Prediction of three-dimensional structures of WRKY proteins associated with abiotic stress response and polysaccharides and flavonoids biosynthesis

The functions of proteins are closely related to their three-dimensional structures [35]. In this study, a total of nine PpWRKYs (PpWRKY20, 39, 75, 76, 90, 92, 115, 122, and 124) were found to be associated with the abiotic stress response and polysaccharides and flavonoids biosynthesis in P. palustre. Subsequently, the three-dimensional structures of the nine PpWRKY proteins were predicted via homology modeling utilizing the SWISS-MODEL tool. The model exhibiting the highest degree of confidence was selected, and the corresponding predictions were presented in Fig. 10.

Fig. 10
figure 10

Prediction of three-dimensional structures of WRKY proteins associated with abiotic stress response and polysaccharides and flavonoids biosynthesis

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Discussion

As one of the largest gene families in plants, WRKY transcription factors are widely involved in plant growth and development, response to adversity stress, and hormone transduction [36, 37]. The identification of the WRKY gene family has been completed in several species, such as Centipedegrass [38], luffa [39], Rheum palmatum [40], etc. However, the WRKY transcription factors have not yet been identified and analyzed at the genome-wide level in P. palustre. In this study, the PpWRKY gene family was analyzed and identified at the genome-wide level, and a total of 133 PpWRKY genes were obtained, which was more than other Lamiaceae species, such as Salvia miltiorrhiza Bunge (69) [41], Scutellaria baicalensis Georgi (77) [42], Prunella vulgaris (23) [43], etc. Phylogenetic analysis showed that the PpWRKY gene family was mainly divided into three groups, Group I, II (IIa, IIb, IIc, IId, IIe, IIf), and III, with Group II having the highest number of members (Fig. 3). It was in line with the results of other species, such as cassava [44], Melastoma dodecandrum [45], Scutellaria baicalensis Georgi [46], and so on. Compared with other species, the number of members in the PpWRKY gene family is relatively large, which may be related to the duplication events of WRKY transcription factors during evolution [47].

Gene duplication is one of the main drivers of the expansion of plant gene family members, and segmental duplication and tandem duplication are the two main forms of gene duplication [48, 49]. It was found that 52 gene duplication events involving 78 ZmWRKY genes were identified in the maize WRKY gene family, and all of them were segmental duplications and did not involve tandem duplications [50]. In Trifolium repens, a total of 124 gene duplication events were identified, including 118 segmental duplications and 6 tandem duplications [51]. In this study, the PpWRKY genes contained 112 segmental duplication genes and 4 tandem duplication genes (Fig. 2). It was similar to the previous results, suggesting that the expansion of the PpWRKY gene family may be primarily driven by segmental duplications. Additionally, gene tandem duplication events frequently occur during plant evolution and are generally regarded as key mechanisms for the expansion and evolution of gene families [52]. After duplication, the repeated WRKY genes may have similar functions and can be simultaneously induced to express by the same stress, forming a complex regulatory network to participate in the regulation of gene expression [47].

The promoter cis-acting elements play an important role in gene transcription, and their activity directly affects the expression and function of genes [53]. Research has shown that MaWRKY80 positively regulates the drought resistance of transgenic Arabidopsis by regulating abscisic acid and redox metabolism [54]. AtWRKY23 regulates root growth by modulating the distribution of auxin [55]. In this study, a variety of cis-acting elements including light-responsive elements, hormone-responsive elements, stress-responsive elements, and development-responsive elements were identified from the promoters of the PpWRKY gene family (Fig. 5), indicating that the PpWRKY gene family may play an important role in responding to and regulating the growth, development, and stress resistance of P. palustre. In addition, based on the analysis of protein-protein interaction networks, we discovered interactions among PpWRKY proteins, suggesting that they may function cooperatively (Fig. 6). Further research is needed to elucidate the mechanisms underlying the role of WRKY protein interactions in the growth, development, and abiotic stress responses of P. palustre.

By constructing a phylogenetic tree with the WRKY gene family of A. thaliana, the function of PpWRKY genes can be further predicted (Fig. 3). Research has shown that AtWRKY8 (AT5G46350) interacts with VQ9 protein, thus participating in regulating plant salt tolerance response [56]. AtWRKY25 (AT2G30250), AtWRKY26 (AT5G07100), and AtWRKY33 (AT2G38470) play important roles in Arabidopsis response to salt stress [57]. In this study, it was found that three PpWRKY genes were in the same branch as AtWRKY2, AtWRKY26, and AtWRKY33, among which PpWRKY72 showed significant changes in expression levels after salt stress (Fig. 8C). AtWRKY40 (AT1G80840) is involved in Arabidopsis drought stress response by regulating plant antioxidant and osmotic regulation abilities [58, 59]. AtWRKY46 (AT2G400), AtWRKY54 (AT2G40750), and AtWRKY70 (AT3G500) inhibit the expression of drought response genes, reducing plant drought resistance [60]. In P. palustre, it was found that PpWRKY53 and PpWRKY121 in the same branch as AtWRKY40 showed significant changes in expression levels after PEG stress (Fig. 8D). AtWRKY33 (AT2G38470) responds to cadmium toxicity by directly activating ATL31 transcription, positively regulating Arabidopsis tolerance to cadmium stress [61]. AtWRKY18 (AT4G31800), AtWRKY40 (AT1G80840), and AtWRKY60 (AT2G25000) enhance Arabidopsis tolerance to cadmium by inhibiting the transcription of genes encoding hydrogen sulfide (H2S) synthesis enzymes [62]. PpWRKY81 in P. palustre in the same branch as AtWRKY33, and PpWRKY5, PpWRKY53, PpWRKY60, PpWRKY121 in the same branch as AtWRKY18, AtWRKY40, and AtWRKY60 exhibited significant changes in expression levels after Cd stress (Fig. 8A). In conclusion, it was speculated that these PpWRKYs might play an important role in the regulation of abiotic stress response in P. palustre.

Polysaccharides and flavonoids are important components of P. palustre. The WRKY genes involved in the biosynthesis and metabolism of polysaccharides and flavonoids in P. palustre are excavated to provide important candidate genes for the research on the biosynthesis and regulation mechanisms of polysaccharides and flavonoids in P. palustre (Fig. 7B, C). Research has shown that OsWRKY13 induces the expression of CHS encoding chalcone synthase, which functions on a branch of the phenylpropanoid pathway involved in flavonoid biosynthesis [63, 64]. The WRKY transcription factor SUSIBA2 in barley activates the activity of starch synthase genes by binding to the SURE element in the iso1 promoter [65, 66]. AtWRKY20 functions as a transcriptional activator of the ApL3 promoter and regulates the expression of ApL3 induced by sucrose or osmoticum in A. thaliana [67]. AtWRKY23 is involved in regulating flavonoid biosynthesis, and its expression is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway (68). In our study, among the 133 PpWRKY family genes, 129 PpWRKYs were expressed at different development stages of stems and leaves (Fig. 7A). The correlation analysis results further suggested that PpWRKY20, PpWRKY39, PpWRKY75, PpWRKY76, PpWRKY90, PpWRKY115, PpWRKY122, and PpWRKY124, were highly correlated with monosaccharides and flavonoids (Fig. 7B, C). It was indicated that these 8 PpWRKYs may be involved in the regulation of the synthesis of polysaccharides and flavonoids in P. palustre. Meanwhile, we conducted three-dimensional structural predictions for the 9 screened PpWRKYs (Fig. 10), but further research is required to elucidate the roles of these genes in the biosynthesis of polysaccharides and flavonoids in P. palustre.

Conclusions

This study identified 133 PpWRKY gene family members (PpWRKYs) at the whole genome level of P. palustre, which are unevenly distributed on 15 chromosomes. Based on their protein structure and phylogenetic characteristics, the 133 PpWRKYs were divided into 3 subgroups. Segmental duplication events play a crucial role in the expansion of the PpWRKY gene family. Through the transcriptome expression data analysis, the candidate genes potentially involved in the growth and development, abiotic stress response, polysaccharide, and flavonoid biosynthesis of P. palustre were preliminarily screened. This study provided a theoretical basis for further understanding the characteristics of the PpWRKY gene family and exploring the biological functions of PpWRKY genes.

Data availability

Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information with the primary accession codes PRJNA777790, PRJNA917426, PRJNA1113799, and PRJNA783728.

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Funding

This study was funded by the Guangxi Key R&D Program Project (GuikeAB24010015), Fund Projects of Central Government in Guidance of Local Science and Technology Development (GuiKeZY22096020), National Natural Science Foundation of China (82260750), and Guangxi Qihuang Scholars Training Program (GXQH202402).

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Author notes

  1. Zhining Chen and Meihua Xu contributed equally to this work.

Authors and Affiliations

  1. Guangxi Key Laboratory of Medicinal Resources Protection and Genetic Improvement/ Guangxi Engineering Research Center of TCM Resource Intelligent Creation, National Center for TCM Inheritance and Innovation, Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China

    Zhining Chen, Meihua Xu, Changqian Quan, Shu Lin, Jingchun Li, Fan Wei & Danfeng Tang

  2. National Engineering Research Center for Southwest Endangered Medicinal Materials Resources Development, Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China

    Meihua Xu, Changqian Quan, Fan Wei & Danfeng Tang

  3. School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing, 210000, China

    Zhining Chen, Jingchun Li & Danfeng Tang

  4. College of Agriculture, Guangxi University, Nanning, 530004, China

    Shu Lin & Danfeng Tang

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  1. Zhining Chen
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  2. Meihua Xu
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  3. Changqian Quan
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Contributions

Zhining Chen, Meihua Xu: Writing-original draft, Data analysis. Changqian Quan, Shu Lin, Jingchun Li: Visualization, Formal analysis. Fan Wei: Supervision, Writing-review & editing. Danfeng Tang: Conceptualization, Writing-review & editing, Funding acquisition. All authors reviewed the manuscript.

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Correspondence to Fan Wei or Danfeng Tang.

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12870_2024_5835_MOESM1_ESM.xlsx

Supplementary Material 1: Table S1: The expression patterns of PpWRKY gene family members in stems and leaves across various growth stages. Table S2: Primers used for RT-qPCR. Table S3: Classification of cis-acting elements of PpWRKY gene family.

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Chen, Z., Xu, M., Quan, C. et al. Genome-wide identification and expression analysis of the WRKY gene family reveal essential roles in abiotic stress responses and polysaccharides and flavonoids biosynthesis in Platostoma palustre (Blume) A. J. Paton. BMC Plant Biol 24, 1122 (2024). https://doi.org/10.1186/s12870-024-05835-6

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BMC Plant Biology

ISSN: 1471-2229

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