Abstract
Background
WRKY transcription factors (TFs) are key regulators of plant stress resistance. Despite their importance, the regulatory mechanisms of WRKYs in Populus trichocarpa under biotic stress remain insufficiently explored. This study presents a comprehensive bioinformatic characterization of the WRKY gene family in P. trichocarpa.
Methods
The most recent P. trichocarpa genome was employed to identify all WRKY genes in the species. Detailed analyses, including molecular characterization, phylogenetic relationships, gene structure, motif and promoter composition, chromosomal localization, and syntenic relationships, were conducted. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression profiles of PtWRKY genes under insect stress.
Results
A total of 139 WRKY genes were identified, unevenly distributed across 18 chromosomes and classified into three principal groups. Conserved motif analysis showed that all PtWRKY proteins contained motifs 1, 2, and 4, with members of the same subgroup exhibiting highly similar motif architectures. Collinearity analysis identified 91 homologous gene pairs within the PtWRKY family, suggesting potential functional redundancy. Examination of the 2,000 bp upstream promoter regions revealed diverse cis-acting elements associated with light responsiveness, phytohormone signaling, and stress regulation. Moreover, several PtWRKY genes responded to feeding stress caused by Hyphantria cunea.
Conclusion
Collectively, these findings elucidate the genomic organization and potential regulatory roles of the WRKY gene family in P. trichocarpa, highlighting their involvement in insect-induced defense and offering insights for future agricultural improvement strategies.
Keywords: Populus trichocarpa, WRKY gene family, Genome-wide identification, Hyphantria cunea
Introduction
Herbivorous insects are significant pests in agriculture and forestry, causing substantial economic and ecological losses. In response to herbivory, plants activate signaling pathways that trigger specific defense mechanisms critical for their survival. Extensive research has established transcription factors (TFs) as key regulators of gene expression, playing pivotal roles in plant defense against herbivores (Romani & Moreno, 2021; Jiang et al., 2017; Phukan, Jeena & Shukla, 2016). By binding to cis-regulatory elements in the promoter regions of target genes, TFs modulate gene expression and facilitate coordinated crosstalk among signal transduction pathways (Javed & Gao, 2023; Priest, Filichkin & Mockler, 2009; Shrestha, Khan & Dey, 2018), ultimately strengthening plant resistance to stress. Given their role as master regulators of defense-related genes, TFs present promising candidates for genetic engineering applications (Chen et al., 2017). Consequently, understanding the mechanistic actions of TFs is vital for advancing insect resistance research.
The WRKY TF was first identified in sweet potato (Ipomoea batatas) (Ishiguro & Nakamura, 1994). Subsequent systematic analysis of the WRKY gene family have been conducted in various plants, including Arabidopsis, rice, and maize (Dong, Chen & Chen, 2003; Ross, Liu & Shen, 2007; Hu et al., 2021). The WRKY family represents one of the largest groups of transcriptional regulators in plants (Mangelsen et al., 2008), characterized by the highly conserved WRKY domain. Each WRKY protein contains one or two conserved domains of approximately 60 amino acids, featuring a highly conserved WRKYGQK heptapeptide at the N-terminus and, in some cases, a zinc finger structure at the C-terminus, either C-X4–5-C-X22–23-HXH (C2H2) or C-X7-C-X23-HXC (C2HC) (Rushton et al., 2010). The WRKY domain exhibits a strong affinity for the conserved W-box (C/TTGACT/C) binding site, which is also present in multiple defense-related genes, highlighting its critical biological functions (Ulker & Somssich, 2004; Yamasaki et al., 2012). As versatile regulators, WRKY genes are involved in nearly all aspects of plant life, from growth and development to tolerance of various abiotic and biotic stresses (Wang et al., 2023). Overexpression of the PtoWRKY60 gene in poplar plants can significantly enhance their resistance to Dothiorella gregaria Sacc (Ye et al., 2014). However, this also leads to adverse phenotypes such as slowed growth, premature leaf senescence and shedding. Several studies have highlighted the role of WRKY genes in herbivore-induced plant defense. For instance, silencing the SlWRKY70 gene in tomato (Lycopersicon esculentum) inhibited the Mi-1 gene-mediated anti-aphid pathway, increasing the susceptibility of mutant plants to aphids (Atamian, Eulgem & Kaloshian, 2012). Overexpression of the OsWRKY89 gene enhanced rice resistance to the white-backed planthopper (Wang et al., 2007). However, despite these advancements, research on the defense mechanisms of PtWRKY genes targeting herbivorous insects remains limited, revealing a significant gap in current knowledge.
Populus trichocarpa serves as a model species for woody plants, with its genome sequence widely utilized in genetic studies since its sequencing and publication in 2006 (Tuskan et al., 2006). It is a key model plant for research on stress resistance in Populus due to its rapid growth, well-defined genetic background, and ease of genetic transformation (Liu et al., 2022; Xu et al., 2021). Although the WRKY gene family in P. trichocarpa has been previously analyzed, with subgroup III gene expression patterns studied under cold, drought, salinity, and salicylic acid stresses (He et al., 2012), a comprehensive analysis of PtWRKY genes under insect stress remains unexplored. In this study, the most recent P. trichocarpa genome was employed to identify all WRKY genes in the species. Detailed analyses, including molecular characterization, phylogenetic relationships, gene structure, motif and promoter composition, chromosomal localization, and syntenic relationships, were conducted. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression profiles of PtWRKY genes under insect stress. This research enhances the functional characterization (physicochemical properties, domains, evolutionary relationships, regulatory elements, and expression patterns under insect stress conditions). of PtWRKY genes and identifies key candidate genes for improving poplar resistance to pest stress.
Materials and Methods
Plant and insect materials and stress treatment
Three 2-month-old P. trichocarpa seedlings with consistent growth were selected for the experiment. The plants were randomly assigned to either a control or a treatment group. For each group, three independent plants were established as biological replicates. From the same node position (the 3rd–5th nodes) of each plant, three healthy and uniformly growing leaves were selected. Hyphantria cunea were provided by the Chinese Academy of Forestry Sciences, were incubated in an artificial climate chamber (L:D = 14:10; temperature 25 ± 1 °C; humidity 70 ± 5%) until the second instar. Prior to treatment, H. cunea larvae were subjected to a starvation protocol, with nine larvae starved for 12 h and placed evenly on the leaves. P. trichocarpa samples were collected 12 h after treatment. To ensure independence across time points, each sampling event used entirely new plants. All experiments were performed in triplicate, and the collected samples were immediately frozen in liquid nitrogen and stored at −80 °C for total RNA extraction. The samples collected at 0 h post-treatment served as the control group.
Identification of PtWRKY genes
Genomic sequences, coding sequences, and protein sequences of P. trichocarpa were retrieved from the National Center for Biotechnology Information (GCF_000002775.5) database (https://www.ncbi.nlm.nih.gov). The 71 WRKY protein sequences from A. thaliana were downloaded from the TAIR (GCF_000001735.4) website (https://www.arabidopsis.org) as reference sequences. Using the WRKY protein sequence from A. thaliana as a query, the BLAST program was employed to compare sequences in the P. trichocarpa genome. Duplicates were merged and removed to obtain the candidate WRKY TF protein sequences of P. trichocarpa. Further identification of PtWRKY genes was carried out using the NCBI CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) for protein domain analysis, ultimately leading to the identification of all WRKY genes in P. trichocarpa. Basic protein properties, including amino acid length, molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY), were predicted using the ExPASy ProtParam tool (https://web.expasy.org/protparam/). The subcellular localization of PtWRKY proteins was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) (Chou & Shen, 2010).
Construction of phylogenetic tree
Phylogenetic trees were constructed using the neighbor joining (NJ) method in MEGA11.0 software with a bootstrap value set to 1,000 (Kumar, Stecher & Tamura, 2016). The evolutionary tree was visualized using the iTOL online tool (http://iTOL.embl.de) (Letunic & Bork, 2019).
Visualization of gene structure and conserved motif of WRKY
Conservative motif analysis of the P. trichocarpa WRKY family protein sequences was conducted using the MEME tool (https://meme-suite.org/meme/tools/meme) (Tang et al., 2008), setting the number of motifs to eight. Based on the P. trichocarpa genome GFF3 annotation file, the gene structure of PtWRKY genes was analyzed and visualized using TBtools software.
Collinearity and chromosomal mapping of WRKY
The DNA sequences of the entire WRKY gene family in P. trichocarpa were mapped across the genome. The distribution of these genes on chromosomes and scaffolds was analyzed using TBtools software. Genome data for A. thaliana, Salix purpurea, and Oryza sativa were downloaded from the Ensembl website, organized, and covariance analysis across species was performed and visualized using TBtools software (Chen et al., 2020).
Predicted co-expression and interaction network
For protein interaction networks, homologous WRKY proteins in Arabidopsis were identified using STRING (https://string-db.org) with a threshold value >0.7. The homologous proteins of the identified interactive P. trichocarpa proteins were determined by reciprocal best BLASTP analysis. The network was then visualized and analyzed using Cytoscape version 3.7.0 (Shannon et al., 2003).
Analysis of cis-regulatory elements
The promoter sequences, extending 2,000 bp upstream of the start codon, were extracted from the P. trichocarpa genome file using TBtools software. Cis-acting elements within the PtWRKY gene promoters were identified using the online tool Plant Care (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) (Lescot et al., 2002).
RNA isolation and RT-qPCR
Total RNA was extracted from P. trichocarpa leaf (the third to fifth healthy and fully expanded leaves) tissues using a plant RNA extraction kit (Tiangen, Beijing, China). The RNA was reverse-transcribed into cDNA using a reverse transcription kit (TaKaRa, Beijing, China). RT-qPCR primers for candidate genes were designed with Primer Premier (version 5.0) (Table 1). Gene expression levels were quantified using the CFX Opus 96 Real-Time PCR system (Bio-Rad, Singapore) and TB Green Premix EX Taq II FAST qPCR (TaKaRa, Beijing, China). The PCR conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, with melting curve conditions at 65 °C for 5 s and 95 °C for 5 s. The poplar actin gene served as the internal reference. The relative expression levels of each gene were calculated using the 2−∆∆Ct method. It includes three cases of biological replication and three cases of technical replicates.
Table 1. The primer sequence information for RT-PCR.
| ID | Gene name | Forward primer sequence (F) | Reverse primer sequence (R) |
|---|---|---|---|
| XM_002297947.4 | PtWRKY2 | TGAAGATCCCACCGTATTTGAG | GTTGTGGAAGTTAAAGAGGGC |
| XM_002298817.4 | PtWRKY3 | TCATCCTAAACCACAACCCAG | TTGGTTAGGCAGAGATGAAGAC |
| XM_002303816.4 | PtWRKY16 | AGTGGAATTGAACCGAGTGAG | CTCTCAGAGTGCCCATTCAT |
| XM_002309030.4 | PtWRKY26 | TCGGGTTTGGAAGCAATAGG | AGGCGGAGAACGGTTGAAGACAC |
| XM_002309150.4 | PtWRKY27 | GATCAATTTGTCTTGGACCCG | GATTGGAAGGATGTGGTAAAGAATG |
| XM_002311293.4 | PtWRKY30 | CAGTTACCCCGAGAAAATCCTC | GTACGATGTCCCCAAGCAG |
| XM_006369831.3 | PtWRKY54 | GTTCCAGCCACCCCTAATTC | TCCTCTGGCTTTTCTGGTTG |
| XM_024583529.2 | PtWRKY76 | GATCCCTACACATTCGAGGTG | AATTCCAGCCATCTAGCGAG |
| XM_024585451.2 | PtWRKY80 | TCAAGTACAATCACAGAACCCC | TTCTTTCAACGAGACCTCCAC |
| XM_024601099.2 | PtWRKY101 | AGCGGATATAGTGGTGCAAC | TGTGGGCTGTTCTTGACTG |
| XM_024604661.2 | PtWRKY107 | GCTCTGAAACGACAAGGAAATG | TTTCCACTTGCCCATCCC |
| XM_024606501.2 | PtWRKY112 | CAGCTTCTATCTCTTCCTCTTCG | GTCATGAAAGCAAATCTCGGC |
| Actin | AATACCCCATTGAGCACGG | ACTCACACCATCACCAGAATC |
Results
Identification and physicochemical property analysis of PtWRKY genes
Conserved domains were identified using NCBI-CDD and Pfam, resulting in the identification of 139 PtWRKY genes. Based on their chromosomal arrangement, these genes were renamed PtWRKY1 through PtWRKY139 (Table 2). Physicochemical property analysis revealed that the length of WRKY proteins ranged from 157 amino acids (PtWRKY18) to 739 amino acids (PtWRKY81), with significant variation in the number of amino acids. The MW varied between 17,992.37 Da (PtWRKY18) and 79,601.83 Da (PtWRKY81). The pI ranged from 4.98 (PtWRKY129) to 9.86 (PtWRKY25). All PtWRKY genes were hydrophilic, with a mean hydrophilicity index ranging from −1.1 to −0.493. Predicted subcellular localization indicated that all 139 PtWRKY genes are located in the nucleus.
Table 2. Physical and chemical properties of PtWRKKY.
| Name | Gene ID | Family | WRKY area | Number of amino acid | Molecular weight/Da | Theoretical pI | Subcellular location |
|---|---|---|---|---|---|---|---|
| PtWRKY1 | rna-XM_002297587.4 | Group II-d | WRKYGQK | 314 | 35,189.87 | 9.45 | Nucleus |
| PtWRKY2 | rna-XM_002297947.4 | Group III | WRKYGQK | 338 | 37966.72 | 5.79 | Nucleus |
| PtWRKY3 | rna-XM_002298817.4 | Group I | WRKYGQK | 557 | 60,421.64 | 6.53 | Nucleus |
| PtWRKY4 | rna-XM_002300084.4 | Group II-c | WRKYGQK | 186 | 21,227.88 | 9.44 | Nucleus |
| PtWRKY5 | rna-XM_002300560.4 | Group I | WRKYGQK | 731 | 78,739.83 | 6.16 | Nucleus |
| PtWRKY6 | rna-XM_002301130.4 | Group II-d | WRKYGQK | 358 | 39,612.68 | 9.51 | Nucleus |
| PtWRKY7 | rna-XM_002301201.4 | Group II-c | WRKYGQK | 203 | 23,424.71 | 7.64 | Nucleus |
| PtWRKY8 | rna-XM_002301341.4 | Group II-c | WRKYGQK | 193 | 21,894.03 | 9.06 | Nucleus |
| PtWRKY9 | rna-XM_002301478.4 | Group II-d | WRKYGQK | 245 | 27,692.54 | 5.71 | Nucleus |
| PtWRKY10 | rna-XM_002301488.4 | Group II-c | WRKYGQK | 317 | 35,402.53 | 6.67 | Nucleus |
| PtWRKY11 | rna-XM_002302034.4 | Group II-d | WRKYGQK | 351 | 38,916.09 | 9.74 | Nucleus |
| PtWRKY12 | rna-XM_002302104.4 | Group II-c | WRKYGQK | 325 | 36,596.37 | 7.65 | Nucleus |
| PtWRKY13 | rna-XM_002302584.4 | Group III | WRKYGQK | 363 | 40,939.24 | 5.82 | Nucleus |
| PtWRKY14 | rna-XM_002302772.4 | Group II-b | WRKYGQK | 506 | 54,489.39 | 7.56 | Nucleus |
| PtWRKY15 | rna-XM_002303574.4 | Group II-e | WRKYGQK | 330 | 37,033.59 | 5.49 | Nucleus |
| PtWRKY16 | rna-XM_002303816.4 | Group II-a | WRKYGQK | 319 | 35,674.03 | 8.49 | Nucleus |
| PtWRKY17 | rna-XM_002304513.4 | Group III | WRKYGQK | 342 | 38,391.23 | 5.25 | Nucleus |
| PtWRKY18 | rna-XM_002304705.4 | Group II-c | WRKYGQK | 157 | 17,992.37 | 9.58 | Nucleus |
| PtWRKY19 | rna-XM_002305235.4 | Group II-e | WRKYGQK | 432 | 47,090.98 | 5.08 | Nucleus |
| PtWRKY20 | rna-XM_002305694.4 | Group II-d | WRKYGQK | 262 | 29,293.51 | 5.56 | Nucleus |
| PtWRKY21 | rna-XM_002306707.4 | Group II-c | WRKYGQK | 322 | 36,311.07 | 7.19 | Nucleus |
| PtWRKY22 | rna-XM_002306787.4 | Group II-d | WRKYGQK | 347 | 38,556.76 | 9.64 | Nucleus |
| PtWRKY23 | rna-XM_002308502.3 | Group II-c | WRKYGQK | 165 | 18,800.72 | 5.48 | Nucleus |
| PtWRKY24 | rna-XM_002308668.3 | Group II-a | WRKYGQK | 320 | 35,415.62 | 8.27 | Nucleus |
| PtWRKY25 | rna-XM_002308962.4 | Group II-d | WRKYGQK | 301 | 32,898.03 | 9.86 | Nucleus |
| PtWRKY26 | rna-XM_002309030.4 | Group II-a | WRKYGQK | 304 | 34,029.43 | 5.52 | Nucleus |
| PtWRKY27 | rna-XM_002309150.4 | Group III | WRKYGQK | 333 | 37,705.54 | 6.04 | Nucleus |
| PtWRKY28 | rna-XM_002310008.4 | Group II-c | WRKYGKK | 233 | 26,378.91 | 9.32 | Nucleus |
| PtWRKY29 | rna-XM_002310086.4 | Group II-d | WRKYGQK | 335 | 36,803.7 | 9.65 | Nucleus |
| PtWRKY30 | rna-XM_002311293.4 | Group II-c | WRKYGQK | 293 | 32,254.6 | 5.85 | Nucleus |
| PtWRKY31 | rna-XM_002312231.4 | Group I | WRKYGQK | 492 | 54,144 | 8.7 | Nucleus |
| PtWRKY32 | rna-XM_002312282.4 | Group II-c | WRKYGQK | 368 | 40,893.57 | 7.06 | Nucleus |
| PtWRKY33 | rna-XM_002314926.4 | Group II-c | WRKYGQK | 374 | 41,381.84 | 6.2 | Nucleus |
| PtWRKY34 | rna-XM_002314988.4 | Group I | WRKYGQK | 499 | 54,536.98 | 8.57 | Nucleus |
| PtWRKY35 | rna-XM_002317361.4 | Group II-d | WRKYGQK | 268 | 30,051.11 | 5.78 | Nucleus |
| PtWRKY36 | rna-XM_002318337.4 | Group III | WRKYGQK | 371 | 41,419.78 | 5.56 | Nucleus |
| PtWRKY37 | rna-XM_002318711.4 | Group II-c | WRKYGQK | 186 | 21,494.98 | 8.9 | Nucleus |
| PtWRKY38 | rna-XM_002319046.4 | Group II-d | WRKYGQK | 354 | 40,094.08 | 9.79 | Nucleus |
| PtWRKY39 | rna-XM_002319843.4 | Group III | WRKYGQK | 324 | 36,719.84 | 5.38 | Nucleus |
| PtWRKY40 | rna-XM_002319923.4 | Group I | WRKYGQK | 591 | 64,943.87 | 7.2 | Nucleus |
| PtWRKY41 | rna-XM_002320124.4 | Group II-c | WRKYGQK | 189 | 21,456.47 | 9.22 | Nucleus |
| PtWRKY42 | rna-XM_002320816.4 | Group III | WRKYGQK | 365 | 41,209.55 | 5.1 | Nucleus |
| PtWRKY43 | rna-XM_002320931.4 | Group II-c | WRKYGQK | 319 | 35,894.11 | 7.06 | Nucleus |
| PtWRKY44 | rna-XM_002322238.4 | Group II-c | WRKYGQK | 178 | 20,390.76 | 9.42 | Nucleus |
| PtWRKY45 | rna-XM_002323601.4 | Group I | WRKYGQK | 579 | 63,971.87 | 6.2 | Nucleus |
| PtWRKY46 | rna-XM_002323803.4 | Group II-b | WRKYGQK | 538 | 58,374.23 | 6.26 | Nucleus |
| PtWRKY47 | rna-XM_002324292.4 | Group II-a | WRKYGQK | 271 | 30,475.4 | 7.59 | Nucleus |
| PtWRKY48 | rna-XM_002324346.4 | Group II-d | WRKYGQK | 338 | 36,823.47 | 9.45 | Nucleus |
| PtWRKY49 | rna-XM_002325212.4 | Group II-d | WRKYGQK | 300 | 33,025.26 | 9.81 | Nucleus |
| PtWRKY50 | rna-XM_002326057.4 | Group I | WRKYGQK | 603 | 66,358.41 | 6.69 | Nucleus |
| PtWRKY51 | rna-XM_006368449.3 | Group II-a | WRKYGQK | 318 | 35,131.43 | 8.85 | Nucleus |
| PtWRKY52 | rna-XM_006368706.3 | Group II-e | WRKYGQK | 325 | 36,713.96 | 5.69 | Nucleus |
| PtWRKY53 | rna-XM_006368806.3 | Group II-c | WRKYGQK | 160 | 18,342.73 | 9.58 | Nucleus |
| PtWRKY54 | rna-XM_006369831.3 | Group II-c | WRKYGQK | 312 | 34,893.44 | 6.31 | Nucleus |
| PtWRKY55 | rna-XM_006371700.3 | Group II-a | WRKYGQK | 320 | 35,569.97 | 9.03 | Nucleus |
| PtWRKY56 | rna-XM_006372308.3 | Group II-e | WRKYGQK | 412 | 45,622.66 | 5.65 | Nucleus |
| PtWRKY57 | rna-XM_006373154.3 | Group II-c | WRKYGQK | 186 | 21,250.89 | 9.61 | Nucleus |
| PtWRKY58 | rna-XM_006375106.3 | Group II-c | WRKYGQK | 228 | 26,409.89 | 6.26 | Nucleus |
| PtWRKY59 | rna-XM_006375289.3 | Group II-e | WRKYGQK | 349 | 38,367.88 | 6.16 | Nucleus |
| PtWRKY60 | rna-XM_006375493.3 | Group I | WRKYGQK | 485 | 53,397.61 | 5.98 | Nucleus |
| PtWRKY61 | rna-XM_006375494.3 | Group I | WRKYGQK | 485 | 53,397.61 | 5.98 | Nucleus |
| PtWRKY62 | rna-XM_006377952.3 | Group I | WRKYGQK | 725 | 78,446.32 | 5.83 | Nucleus |
| PtWRKY63 | rna-XM_006380631.3 | Group II-d | WRKYGQK | 334 | 36,716.62 | 9.65 | Nucleus |
| PtWRKY64 | rna-XM_006381457.3 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY65 | rna-XM_006382750.3 | Group II-d | WRKYGQK | 353 | 39,696.51 | 9.69 | Nucleus |
| PtWRKY66 | rna-XM_006383468.3 | Group II-d | WRKYGQK | 331 | 36,663.4 | 9.38 | Nucleus |
| PtWRKY67 | rna-XM_006386554.3 | Group II-e | WRKYGQK | 354 | 38,846.56 | 6.15 | Nucleus |
| PtWRKY68 | rna-XM_006389516.2 | Group II-b | WRKYGQK | 590 | 64,185.08 | 6.61 | Nucleus |
| PtWRKY69 | rna-XM_024581109.2 | Group II-c | WRKYGQK | 306 | 34,030.86 | 8.16 | Nucleus |
| PtWRKY70 | rna-XM_024581374.2 | Group I | WRKYGQK | 725 | 78,446.32 | 5.83 | Nucleus |
| PtWRKY71 | rna-XM_024581375.2 | Group I | WRKYGQK | 725 | 78,446.32 | 5.83 | Nucleus |
| PtWRKY72 | rna-XM_024581376.2 | Group I | WRKYGQK | 708 | 76,572.32 | 5.92 | Nucleus |
| PtWRKY73 | rna-XM_024581601.2 | Group I | WRKYGQK | 560 | 60,801.21 | 6.97 | Nucleus |
| PtWRKY74 | rna-XM_024581602.2 | Group I | WRKYGQK | 507 | 55,043.94 | 6.94 | Nucleus |
| PtWRKY75 | rna-XM_024583061.2 | Group I | WRKYGQK | 716 | 78,369.07 | 6.62 | Nucleus |
| PtWRKY76 | rna-XM_024583529.2 | Group III | WRKYGQK | 364 | 40,549.27 | 6.09 | Nucleus |
| PtWRKY77 | rna-XM_024584301.2 | Group II-b | WRKYGQK | 502 | 54,065.41 | 6.9 | Nucleus |
| PtWRKY78 | rna-XM_024584519.2 | Group I | WRKYGQK | 485 | 53,397.61 | 5.98 | Nucleus |
| PtWRKY79 | rna-XM_024585445.2 | Group I | WRKYGQK | 739 | 79,601.83 | 6.16 | Nucleus |
| PtWRKY80 | rna-XM_024585451.2 | Group I | WRKYGQK | 739 | 79,601.83 | 6.16 | Nucleus |
| PtWRKY81 | rna-XM_024585457.2 | Group I | WRKYGQK | 739 | 79,601.83 | 6.16 | Nucleus |
| PtWRKY82 | rna-XM_024585466.2 | Group I | WRKYGQK | 732 | 78,906 | 6.23 | Nucleus |
| PtWRKY83 | rna-XM_024585549.2 | Group II-d | WRKYGQK | 361 | 39,868.07 | 9.36 | Nucleus |
| PtWRKY84 | rna-XM_024585550.2 | Group II-d | WRKYGQK | 360 | 39,780.99 | 9.36 | Nucleus |
| PtWRKY85 | rna-XM_024587515.2 | Group I | WRKYGQK | 469 | 51,535.26 | 9.34 | Nucleus |
| PtWRKY86 | rna-XM_024587516.2 | Group I | WRKYGQK | 469 | 51,535.26 | 9.34 | Nucleus |
| PtWRKY87 | rna-XM_024587806.2 | Group I | WRKYGQK | 556 | 60,364.59 | 6.53 | Nucleus |
| PtWRKY88 | rna-XM_024588647.2 | Group I | WRKYGQK | 532 | 58,247.79 | 7.75 | Nucleus |
| PtWRKY89 | rna-XM_024590285.2 | Group I | WRKYGQK | 527 | 57,897.46 | 5.69 | Nucleus |
| PtWRKY90 | rna-XM_024590287.2 | Group I | WRKYGQK | 523 | 57,483.05 | 5.82 | Nucleus |
| PtWRKY91 | rna-XM_024590516.2 | Group II-c | WRKYGQK | 511 | 56,542.75 | 9.13 | Nucleus |
| PtWRKY92 | rna-XM_024590518.2 | Group II-c | WRKYGQK | 422 | 46,295.02 | 9.26 | Nucleus |
| PtWRKY93 | rna-XM_024594046.2 | Group II-d | WRKYGQK | 357 | 39,525.6 | 9.51 | Nucleus |
| PtWRKY94 | rna-XM_024594979.2 | Group II-c | WRKYGQK | 261 | 29,161.58 | 9.18 | Nucleus |
| PtWRKY95 | rna-XM_024596020.2 | Group II-d | WRKYGQK | 245 | 27,692.54 | 5.71 | Nucleus |
| PtWRKY96 | rna-XM_024596958.2 | Group II-d | WRKYGQK | 304 | 34,405.91 | 9.47 | Nucleus |
| PtWRKY97 | rna-XM_024598510.2 | Group I | WRKYGQK | 543 | 59,495.25 | 7.27 | Nucleus |
| PtWRKY98 | rna-XM_024598865.2 | Group II-d | WRKYGQK | 223 | 25,044.07 | 6.08 | Nucleus |
| PtWRKY99 | rna-XM_024600078.2 | Group II-b | WRKYGQK | 602 | 65,078.08 | 6.29 | Nucleus |
| PtWRKY100 | rna-XM_024600511.2 | Group II-d | WRKYGQK | 332 | 36,750.48 | 9.38 | Nucleus |
| PtWRKY101 | rna-XM_024601099.2 | Group II-c | WRKYGKK | 206 | 22,873.15 | 6.06 | Nucleus |
| PtWRKY102 | rna-XM_024602206.2 | Group II-d | WRKYGQK | 347 | 38,556.76 | 9.64 | Nucleus |
| PtWRKY103 | rna-XM_024602795.2 | Group II-e | WRKYGQK | 448 | 48,448.32 | 5.63 | Nucleus |
| PtWRKY104 | rna-XM_024602932.2 | Group I | WRKYGQK | 546 | 60,333.25 | 8.9 | Nucleus |
| PtWRKY105 | rna-XM_024603986.2 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY106 | rna-XM_024603990.2 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY107 | rna-XM_024604661.2 | Group I | WRKYGQK | 522 | 56,833.44 | 6.32 | Nucleus |
| PtWRKY108 | rna-XM_024604662.2 | Group I | WRKYGQK | 518 | 56,419.02 | 6.57 | Nucleus |
| PtWRKY109 | rna-XM_024605234.2 | Group II-c | WRKYGKK | 192 | 21,887.15 | 7.63 | Nucleus |
| PtWRKY110 | rna-XM_024606155.2 | Group II-c | WRKYGQK | 293 | 32,254.6 | 5.85 | Nucleus |
| PtWRKY111 | rna-XM_024606156.2 | Group II-c | WRKYGQK | 293 | 32,254.6 | 5.85 | Nucleus |
| PtWRKY112 | rna-XM_024606501.2 | Group II-c | WRKYGQK | 318 | 35,270.42 | 7.01 | Nucleus |
| PtWRKY113 | rna-XM_024609281.2 | Group II-d | WRKYGQK | 313 | 35,033.68 | 9.39 | Nucleus |
| PtWRKY114 | rna-XM_024610899.2 | Group II-b | WRKYGQK | 523 | 57,025.65 | 6.09 | Nucleus |
| PtWRKY115 | rna-XM_024611133.2 | Group II-c | WRKYGQK | 293 | 32,595.97 | 6.4 | Nucleus |
| PtWRKY116 | rna-XM_024611531.2 | Group II-b | WRKYGQK | 593 | 64,574.49 | 6.61 | Nucleus |
| PtWRKY117 | rna-XM_024611532.2 | Group II-b | WRKYGQK | 592 | 64,446.32 | 6.48 | Nucleus |
| PtWRKY118 | rna-XM_024611533.1 | Group II-b | WRKYGQK | 589 | 64,056.91 | 6.48 | Nucleus |
| PtWRKY119 | rna-XM_052445140.1 | Group II-e | WRKYGQK | 461 | 49,731.65 | 5.71 | Nucleus |
| PtWRKY120 | rna-XM_052445501.1 | Group I | WRKYGQK | 541 | 59,246.01 | 7.95 | Nucleus |
| PtWRKY121 | rna-XM_052445816.1 | Group II-c | WRKYGQK | 185 | 21,338.79 | 8.68 | Nucleus |
| PtWRKY122 | rna-XM_052446276.1 | Group III | WRKYGQK | 324 | 36,719.84 | 5.38 | Nucleus |
| PtWRKY123 | rna-XM_052447277.1 | Group II-d | WRKYGQK | 262 | 29,899.11 | 5.26 | Nucleus |
| PtWRKY124 | rna-XM_052447278.1 | Group II-d | WRKYGQK | 235 | 26,838.72 | 5.07 | Nucleus |
| PtWRKY125 | rna-XM_052447365.1 | Group II-b | WRKYGQK | 636 | 68,314.18 | 6.49 | Nucleus |
| PtWRKY126 | rna-XM_052449326.1 | Group III | WRKYGQK | 381 | 41,276.09 | 6.1 | Nucleus |
| PtWRKY127 | rna-XM_052450880.1 | Group II-d | WRKYGQK | 245 | 27,917.09 | 5.96 | Nucleus |
| PtWRKY128 | rna-XM_052450881.1 | Group II-d | WRKYGQK | 245 | 27,917.09 | 5.96 | Nucleus |
| PtWRKY129 | rna-XM_052450883.1 | Group II-d | 209 | 23,823.39 | 4.98 | Nucleus | |
| PtWRKY130 | rna-XM_052452687.1 | Group II-c | WRKYGQK | 232 | 26,308.64 | 9.08 | Nucleus |
| PtWRKY131 | rna-XM_052452688.1 | Group II-c | WRKYGQK | 232 | 26,308.64 | 9.08 | Nucleus |
| PtWRKY132 | rna-XM_052453559.1 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY133 | rna-XM_052453560.1 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY134 | rna-XM_052453561.1 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY135 | rna-XM_052453562.1 | Group I | WRKYGQK | 475 | 51,973.49 | 9.12 | Nucleus |
| PtWRKY136 | rna-XM_052454988.1 | Group I | WRKYGQK | 731 | 78,739.83 | 6.16 | Nucleus |
| PtWRKY137 | rna-XM_052455199.1 | Group II-c | WRKYGQK | 293 | 32,254.6 | 5.85 | Nucleus |
| PtWRKY138 | rna-XM_052455200.1 | Group II-c | WRKYGQK | 293 | 32,254.6 | 5.85 | Nucleus |
| PtWRKY139 | rna-XM_052456799.1 | Group II-c | WRKYGQK | 293 | 32,595.97 | 6.4 | Nucleus |
Phylogenetic analysis and multiple sequence alignment of PtWRKY genes
Phylogenetic analysis was conducted using the 139 WRKY protein sequences from P. trichocarpa and 71 known WRKY protein sequences from A. thaliana. The analysis revealed that the 139 P. trichocarpa WRKY TFs could be classified into three major groups (Fig. 1). Group I contained 40 WRKY proteins, group II contained 88 WRKY proteins, and group III contained 10 WRKY proteins. Group II was further subdivided into five subclasses: IIa (six members), IIb (10 members), IIc (36 members), IId (28 members), and IIe (eight members). Based on evolutionary relationships, the WRKY family in higher plants can be divided into three subclasses: IIa + IIb, IIc, and IId + IIe, consistent with the phylogenetic tree results. Multiple sequence alignment (Table 2) showed that 135 of the 139 PtWRKY proteins contained the signature WRKYGQK conserved domain. Four proteins deviated: PtWRKY129 (subgroup IIc) lacked the WRKY domain, while PtWRKY28, PtWRKY101, and PtWRKY109 exhibited mutations in the conserved domain, replacing WRKYGQK with WRKYGKK.
Figure 1. Phylogenetic tree of WRKY proteins.
Conserved motif and exon–intron structure analyses of PtWRKY genes
To investigate the structural diversity of the PtWRKY genes, a rootless evolutionary tree was constructed to analyze conserved motifs and gene structures (Fig. 2). Gene structure analysis revealed that all PtWRKY genes contain introns, with the number of exons ranging from two (PtWRKY26, PtWRKY18, PtWRKY53, PtWRKY57, PtWRKY4, PtWRKY44, PtWRKY8, PtWRKY37, PtWRKY121, PtWRKY41) to six (PtWRKY73, PtWRKY3, PtWRKY87, PtWRKY68, PtWRKY119, PtWRKY118, PtWRKY117, PtWRKY99, PtWRKY14, PtWRKY77, PtWRKY125). The number of introns varied between one and five in the PtWRKY genes. Notably, most PtWRKY genes within the same group exhibited similar exon-intron structures (Figs. 2B and 2C).
Figure 2. PtWRKY conserved motifs and gene structure.
To further explore the structural features and conduct optimal functional analyses of PtWRKY proteins, conserved motifs in the 139 PtWRKY proteins were examined using the MEME online software. This analysis identified eight highly correlated conserved motifs (Fig. 3). The frequency with which each motif occurred within WRKY proteins indicates its significance in the sequence. Notably, motifs 1 and 3 correspond to the conserved WRKYGQK heptapeptide segment. All PtWRKY proteins in the family possess motifs 1, 2, and 4, which represent the core conserved domains of the PtWRKY genes. These motifs play a critical role in maintaining the fundamental characteristics and functional integrity of the gene family.
Figure 3. Conserved motifs of the PtWRKY protein.
Collinearity analysis of WRKY gene family members in P. trichocarpa
During evolution, segmental duplication, whole-genome duplication (WGD), and tandem duplications have played key roles in gene family expansion in plants. In the case of PtWRKY genes, intra-species collinearity analysis revealed fourteen pairs of tandem duplication events, primarily located on chromosomes Chr01, Chr02, Chr06, Chr11, Chr13, Chr14, and Chr18. Evolutionary analysis of the 139 PtWRKY genes identified 91 genes originating from WGD events (Fig. 4), suggesting that WGD was the major contributor to the expansion of the WRKY gene family in Populus.
Figure 4. Distribution of WRKY genes.
To further investigate the phylogenetic relationship of WRKY genes between P. trichocarpa and three other species, interspecific collinearity analysis was performed (Fig. 5). The numbers of orthologous WRKY pairs between P. trichocarpa and S. purpurea, A. thaliana, and O. sativa were 235, 123, and 75, respectively. These results indicate stronger collinearity between P. trichocarpa and S. purpurea, followed by A. thaliana, and finally O. sativa.
Figure 5. Synteny analysis of WRKY genes between P. trichocarpa and three representative plant species.
Chromosome mapping of PtWRKY genes
Using TBtools software, the PtWRKY genes were mapped to P. trichocarpa chromosomes (Fig. 6). The results revealed that these genes are distributed across 18 chromosomes (excluding Chr09), though the distribution and density across chromosomes are uneven. Specifically, the highest number of genes (18) is located on Chr01, while the lowest (only two genes) is found on Chr19. These results confirm the uneven distribution and density of PtWRKY genes across the chromosomes, despite their presence on 18 chromosomes.
Figure 6. Chromosomal location of the WRKY gene family in P. trichocarpa.
Prediction of co-regulatory and interaction networks of PtWRKYs
WRKY TFs represent one of the largest families of transcriptional regulators in plants, integral to signaling networks that modulate various plant processes. To explore the potential regulatory networks among PtWRKY genes, co-expression patterns for the 139 PtWRKY proteins were predicted using the STRING protein-protein interaction database, revealing that 32 genes are involved in interactions (Fig. 7). Notably, PtWRKY55 and PtWRKY122 occupy central positions in the network, displaying extensive connections with numerous other PtWRKY proteins. In contrast, peripheral nodes such as PtWRKY94, PtWRKY131, and PtWRKY129 exhibit relatively fewer connections. These results suggest that PtWRKY55 and PtWRKY122 likely play pivotal roles in regulating the PtWRKY protein network, potentially acting as hub regulators that mediate interactions among a wide range of WRKY family members to coordinate various plant biological processes.
Figure 7. Protein-protein interaction (PPI) network of important genes.
Cis-acting elements analysis of WRKY gene family members in P. trichocarpa
To further investigate the transcriptional regulation and potential functions of PtWRKY genes, PlantCARE was used to predict the cis-acting elements in their promoters. The analysis revealed that, in addition to promoter-related elements and WRKY binding site motifs, three types of cis-regulatory elements were highly concentrated in the PtWRKY gene promoters: light-responsive elements, plant hormone response elements, and environmental stress response-related elements (Fig. 8). Among these, the environmental stress-related elements included low-temperature (LTR), drought (MBS), defense and stress (Tc-rich repeats), and anaerobic induction (ARE) response elements. Light-responsive elements, such as Box4 and G-box, were the most abundant, with all 139 PtWRKY genes containing these motifs. Plant hormone response elements included methyl jasmonate-responsive elements (CGTCA-Motif and TGACG-Motif), abscisic acid-responsive elements (ABRE), auxin responsiveness (AuxRR-core), salicylic acid-responsive elements (TCA-element), and gibberellin-responsive elements (p-box and GARE-motif). These results suggest that the majority of cis-acting elements in PtWRKY genes are associated with stress responses. Analyzing these elements can provide valuable insights into how PtWRKY genes mediate stress responses, particularly under biotic stress conditions.
Figure 8. Cis-acting elements of the Pt WRKY gene family.
Expression analysis of PtWRKY genes under H. cunea feeding
Jasmonic acid (JA) and salicylic acid (SA) are key regulators of plant defense mechanisms against insect herbivores, while abscisic acid (ABA) also plays a role in responding to various stresses, including biotic stresses induced by insects. Based on the central roles of JA, SA, and ABA in plant defense against herbivorous insects, we analyzed the 2,000-bp promoter sequences of the 139 PtWRKY genes using PlantCARE. We specifically prioritized genes containing cis-acting elements responsive to all three hormones, as these genes are likely regulated by multi-hormonal signals and involved in the defense of P. trichocarpa against H. cunea. (Fig. 9). To explore the role of the WRKY gene family in plant defense against H. cunea, the expression of 12 PtWRKY genes was analyzed using RT-qPCR (Fig. 10). Differences between the H. cunea feeding treatment and the control group were assessed using Test statistics (t-values) (* 0.01< p < 0.05; **p < 0.01). Except for PtWRKY2, PtWRK3 and PtWRKY101, which exhibited significant differences, all the other genes showed extremely significant differences (Table 3). The results showed varied expression patterns, which may be attributed to their distinct regulatory functions or the complex interactions of TFs. After H. cunea feeding, the expression levels of PtWRKY2 and PtWRKY3 did not change significantly. In contrast, PtWRKY16 and PtWRKY76 were upregulated in response to insect feeding compared to the control (Fig. 10). The expression of other genes, including, PtWRKY26, PtWRKY80, PtWRKY107, and PtWRKY112, was significantly downregulated following insect feeding.
Figure 9. Plants of Populus trichocar, Hyphantria cunea larvae feeding leaves.
Figure 10. Relative expression levels of all WRKYs under H.cunea treatments.
Table 3. Test statistics.
| Gene | T-value | p-value |
|---|---|---|
| PtWRKY2 | 3.826 | 0.019 |
| PtWRKY3 | 4.011 | 0.016 |
| PtWRKY16 | 6.61 | 0.003 |
| PtWRKY26 | 30.575 | 0.000 |
| PtWRKY27 | 5.846 | 0.004 |
| PtWRKY30 | 9.420 | 0.002 |
| PtWRKY54 | 5.597 | 0.005 |
| PtWRKY76 | −25.022 | 0.000 |
| PtWRKY80 | 11.753 | 0.000 |
| PtWRKY101 | 3.933 | 0.017 |
| PtWRKY107 | 8.417 | 0.001 |
| PtWRKY112 | 5.623 | 0.005 |
Discussion
WRKY TFs are critical regulators, forming one of the largest families of transcriptional regulators with broad involvement in biotic stress responses. P. trichocarpa possesses a notably higher number of WRKY family members compared to several other plant species, including A. thaliana (Dong, Chen & Chen, 2003), G. hirsutum (Dou et al., 2014), Nelumbo nucifera (Li et al., 2019), Prunus persica (Chen et al., 2016), and Ananas comosus (Xie et al., 2018). This variation is likely attributed to P. trichocarpa’s large genome size and the high frequency of repeat events. Using genome data, 139 PtWRKY genes were identified and subjected to comprehensive bioinformatics analysis. Evolutionary analysis classified the PtWRKY genes into three primary groups, with group II further divided into five subgroups (Fig. 1), consistent with classifications observed in other plant species (Huang et al., 2012; Liu et al., 2017; Yang et al., 2015).
Structural analysis revealed that the amino acid sequences of most PtWRKY genes contained the conserved heptapeptide sequence (WRKYGQK), although a few exhibited heptapeptide variants (WRKYGKK, WRAYGGK, and WKKHGEK). These mutations could potentially result in functional changes. In this study, the conserved motif WRKYGQK in the PtWRKY28, PtWRKY101, and PtWRKY109 proteins, belonging to group IIc, was altered to WRKYGKK. Notably, the tobacco (Nicotiana tabacum) NtWRKY12, which contains a WRKYGKK motif, recognizes a downstream binding sequence (TTTTCCAC) that deviates significantly from the W-box (van Verk et al., 2008). Similarly, GmWRKY6 and GmWRKY21 in soybean (Zhou et al., 2008), also containing the WRKYGKK motif, exhibit abnormal binding to the W-box. It is hypothesized that these PtWRKY heptapeptide variants of P. trichocarpa may impair the WRKY protein’s ability to bind to DNA, or potentially enable recognition of novel motifs, leading to the emergence of new functions.
The creation of introns is a significant event in genomic evolution, contributing to species adaptation over time (Zhou et al., 2008; Bong-Seok & Choi, 2015; Patthy, 1999). Gene structure plays a pivotal role in the evolution of multigene families (Penny et al., 2009; Gaba et al., 2023). The number of introns influences plant responses to different developmental stages and environmental stimuli (Ohta, 2013; Liu et al., 2023). In this study, gene structure analysis of PtWRKY genes was integrated with phylogenetic evolution results. Evolutionary studies often classify proteins based on gene structure and motif arrangement relationships (Wiegmann et al., 2019; Hwarari et al., 2023). In this context, motifs 1, 2, and 4, which contain the WRKY domain, are identified as essential components of PtWRKY proteins. Additionally, PtWRKY proteins were categorized based on gene structure and motif configurations. The PtWRKY genes varied in exon count, ranging from two to six exons, and the exon-intron arrangements were consistent within each subfamily. The conserved structural motifs in PtWRKY proteins within the same subgroup were largely identical, suggesting that these proteins are relatively conserved during evolution. However, differences in the conserved motifs between subgroups indicate functional differentiation of PtWRKY proteins in each group.
A significant number of PtWRKY genes were located in duplicated genomic regions, highlighting the importance of large-scale duplication events in the expansion of the PtWRKY gene family. Ninety-one collinearity PtWRKY gene pairs were identified in the P. trichocarpa genome, emphasizing the role of segmental duplication in the expansion of these genes. Evolutionary analysis also revealed substantial synteny between P. trichocarpa, A. thaliana, O. sativa, and S. purpurea, indicating a shared ancestry prior to the divergence of these lineages. Interspecies collinearity analysis further showed that more WRKY gene pairs are present in P. trichocarpa and S. purpurea, suggesting closer genetic similarities between these two tree species. Consequently, the PtWRKY gene family is more closely related to S. purpurea, followed by A. thaliana, while O. sativa is the most distantly related. These findings collectively suggest that the WRKY gene family has undergone varying degrees of expansion across different plant species, aiding their adaptation to diverse environments.
In response to stress, plants adapt to adverse environments through signal transduction and molecular regulation mechanisms. TFs play a critical role in signal transduction by activating or inhibiting the transcription of downstream genes, specifically binding to gene promoter regions, thus modulating the expression of related functional genes (Schwechheimer, Zourelidou & Bevan, 1998). WRKY proteins bind to W-box elements ((C/T)TGAC(T/C)) or other cis-acting elements in the promoter regions of target genes, subsequently regulating the expression of downstream genes (Sun et al., 2003). Analysis of the PtWRKY gene promoters revealed several cis-acting elements responsive to plant hormones and various stresses, including W-box (WRKY TF-binding element), ABRE, ARE (auxin response element), SARE, and MeJARE. These findings suggest that PtWRKY genes may encode key regulators in P. trichocarpa’s stress response mechanisms, mediated through plant hormone pathways.
WRKY proteins are essential in mitigating stress-induced damage and enhancing stress tolerance by activating defense-related genes via signal transduction pathways. These pathways involve key hormones such as ABA (Lim et al., 2022; Liu et al., 2015; Rushton et al., 2012), JA (Hou et al., 2019; Huang et al., 2022; Nguyen, Goossens & Lacchini, 2022), and SA (Cui et al., 2020; van Verk, Bol & Linthorst, 2011). To investigate whether PtWRKY genes are involved in P. trichocarpa defense against the insect herbivore H. cunea via these hormonal pathways, we analyzed the promoter regions of all 139 PtWRKY genes sing PlantCARE. The analysis revealed that a significant subset harbors cis-acting elements responsive to JA, SA, and ABA, providing basics that their expression may be modulated by these defense-related signals. In this study, the analysis of the promoter region of the PtWRKY genes revealed that a part of genes contained cis-acting elements responsive to JA, SA, and ABA. This is consistent with the established role of JA in anti-herbivore defense—where it can be upregulated by WRKY proteins, JA is upregulated by WRKY3 and WRKY6 in Nattemata during insect attacks (Skibbe et al., 2008). BpWRKY6 expression was induced by JA treatment, and the JA content of BpWRKY6-overexpressing birch was also greater than that of the control. Furthermore, Y1H, EMSA, LUC, and ChIP–qPCR results verified that BpWRKY6 can bind to the promoters of JA synthesis genes to promote their expression (Xie et al., 2025). With the documented interplay between SA (Lortzing et al., 2019), ABA (Dinh, Baldwin & Galis, 2013), and JA (Bruce & Pickett, 2007) in enhancing plant resistance.
In this study, the expression levels of 12 PtWRKY genes were analyzed following 12 h of exposure to H. cunea feeding. Notably, PtWRKY16and PtWRKY76 were upregulated in response to insect feeding, compared to the control. Other WRKY genes exhibited low expression after 12 h of exposure to H. cunea. This divergence in expression profiles occurred despite all candidate genes containing cis-elements for JA, SA, and/or ABA, suggesting they are subject to distinct hormonal regulation—where specific signals may dominate or be suppressed—during the early defense response. Several key candidate PtWRKY genes were identified in this study, which could have practical applications in enhancing insect resistance in P. trichocarpa in the field. Further experimentation involving transgenic plants that either silence or overexpress these candidate WRKY genes is necessary to directly identify the transcriptional targets of key WRKY genes. This will provide deeper insights into their roles in P. trichocarpa’s defense responses to H. cunea.
Conclusions
This study identified a total of 139 WRKY gene family members in P. trichocarpa. These genes were classified into five subgroups based on phylogenetic relationships and primarily contained conserved Motifs 1, 2, and 4. The cis-acting elements in the promoters of these WRKY genes were associated with hormone signaling (JA, SA, ABA, etc.), light response, and defense and stress responses. Collinearity analysis revealed a stronger WRKY gene collinearity between P. trichocarpa and S. purpurea, highlighting conserved WRKY evolution within the Salicaceae family. Following H. cunea feeding, PtWRKY16 and PtWRKY76 were upregulated, indicating their role in biotic stress. In summary, this study provides valuable insights into the functional characteristics of PtWRKY genes and contributes to the identification of key stress response genes involved in P. trichocarpa’s defense against H. cunea in this field.
Supplemental Information
Acknowledgments
Our gratitude extends to the College of Forestry and Grassland at Jilin Agriculture University for providing the experimental sites and equipment.
Funding Statement
We received funding from the Technological Innovation 2030-Major Project of Agricultural Biological Breeding (2022ZD0401504). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Contributor Information
Xinxin Zhang, Email: zxx2023@163.com.
Xiyang Zhao, Email: xiyangz@jlau.edu.cn.
Additional Information and Declarations
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Yue Sun conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the article, and approved the final draft.
Hanxi Li analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Yao Chi analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Yanlin Liu analyzed the data, prepared figures and/or tables, and approved the final draft.
Xinxin Zhang conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
Xiyang Zhao analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Data Availability
The following information was supplied regarding data availability:
The data is available in the Supplemental File and at NCBI: GCF_000002775.5.
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Supplementary Materials
Data Availability Statement
The following information was supplied regarding data availability:
The data is available in the Supplemental File and at NCBI: GCF_000002775.5.