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Article

Comparative Identification of LsWRKY Transcription Factors and Transcriptional Response to Abiotic and Biotic Stresses in Lagenaria siceraria

by
Han Jin
1,*,
Shuoshuo Wang
2,
Wenli Li
2,
Shujing Tan
2 and
Yan Zhao
2
1
School of Pharmaceutical Science and Food Engineering, Liaocheng University, Liaocheng 252000, China
2
College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1192; https://doi.org/10.3390/horticulturae11101192
Submission received: 8 April 2025 / Revised: 13 September 2025 / Accepted: 16 September 2025 / Published: 3 October 2025
(This article belongs to the Special Issue Genetics and Breeding of Cucurbitaceae Crops)
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Abstract

Lagenaria siceraria is an essential horticultural and medicinal crop that is used for its edible fruits and ornamental purposes. WRKY transcription factors have been extensively studied in plant responses to environmental stress; however, there is limited information on their specific functions in L. siceraria. In this study, 51 LsWRKY genes were identified in the L. siceraria genome. The 51 LsWRKYs were divided into classes I, II, and III based on evolutionary analysis. Members of each class have similar conserved motifs and exon-intron structures, and promoter analysis helped identify many cis-regulatory elements associated with growth, hormones, and stress responses. GO terms and KEGG analyses indicated the potential roles of LsWRKY in the regulation of bottle gourd development and acclimation to various environmental stressors. Significant differences in LsWRKY expression were observed between different tissues. The results of RNA-seq and qRT-PCR showed that LsWRKYs were expressed in a tissue- and development-specific manner under normal growth conditions. LsWRKY abundance showed a clear pattern of change related to stress when L. siceraria was exposed to unfavorable environmental conditions. This study provides new insights into the role of LsWRKYs in the growth and stress responses of cucurbits.

1. Introduction

Transcription factors (TFs) selectively bind to specific sequences in gene promoter regions, thereby regulating the expression of related genes [1,2]. TFs are involved in critical signal transduction pathways that respond to abiotic and biotic stressors during plant growth [3]. Current research on TFs has focused predominantly on WRKY, MYB, NAC, and bHLH, which are closely related to plant adversity responses [4,5,6,7]. WRKY genes, a class of TFs unique to higher plants, are among the most important TF families implicated in plant stress responses [8,9,10,11].
Since the isolation of the first plant WRKY TF from sweet potato (Ipomoea batatas) in 1994, a growing number of WRKY TFs have been discovered and characterized in plants, which are now recognized as key regulators of plant growth, development, and stress responses [12]. WRKY TFs are best characterized by a 60-amino-acid DNA-binding domain with a highly conserved WRKYGQK heptapeptide at the N terminus and a conserved zinc-finger motif at the C terminus [13]. This structural domain can specifically interact with DNA sequences known as the W-box cis-regulatory element to regulate gene expression [14]. Based on the number of WRKY structural domains and zinc-finger structural domain characterization, the WRKY gene family is divided into three classes. Class I contains two WRKY structural domains, and classes II and III contain one WRKY structural domain with C2H2- and C2HC-type zinc-finger structures, respectively [13]. Members of Class III can participate in regulating the trade-off between plant growth and drought resistance through brassinosteroid-mediated signal transduction [15,16,17]. Class II can be divided into five subgroups (IIa, IIb, IIc, IId, and IIe). AtWRKY7, AtWRKY11, AtWRKY15, AtWRKY17, AtWRKY21, and AtWRKY39 belong to Class IId in Arabidopsis and play dual roles in the drought stress response, growth, and drought tolerance [18]. Members of Class IId play key roles in the regulation of plant growth and drought tolerance [19]. In addition, some WRKY TFs in rice have been categorized as Class IV because of their structural domain residues [20]. Amino acid substitutions in the WRKYGQK structural domain are present in many plant WRKY genes, including WRKYGKK, WRKYGEK, WSKYEQK, and WRKYSEK [21,22]. Alterations in the WRKYGQK sequence affect the DNA-binding affinity, and some mutants lack DNA-binding ability [11].
WRKY TFs are critical factors in multigene regulatory systems that respond to a wide range of environmental stresses, including salt, heat, cold, drought, and pathogens. ZmWRKY114 negatively regulates salt stress [23]. SlWRKY8 activates W-box-dependent transcription in tomato, and overexpression (OE) of SlWRKY8 improves tomato resistance to pathogens, regulates increased transcript levels of SlPR1a1 and SlPR7, and confers resistance to drought and salt stress [24]. OEOsWRKY10 promotes the accumulation of reactive oxygen species (ROS) in chloroplasts and plasmodesmata and induces the expression of heat shock TFs and protein genes [25]. OsWRKY11 binds to the HSP101 promoter and improves heat and drought tolerance in rice [26]. OECsWRKY46 regulates cold-stress gene expression by mediating the abscisic acid (ABA) signaling pathway and enhancing cold resistance in plants [27]. ZmWRKY79 positively regulates drought resistance by increasing ABA biosynthesis [28]. GmWRKY16 mediates the ABA signaling pathway to improve soybean tolerance to drought and salt stress by inducing the expression of WRKY, stress-related markers, signaling, and responsive genes [29,30]. AtWRKY52 (RRS1) contains the TIR-NBS-LRR and WRKY structural domains and mediates resistance to bacterial pathogens [31]. WRKY TFs are involved in the regulation of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), ethylene (ET), and brassinosteroid (BR) signaling, and function as a regulator and balancer in the interplay of these signaling pathways [32,33,34]. OENtWRKY50 resulted in altered SA and JA content, increased defense-related gene expression, and enhanced tobacco resistance to R. solanacearum [35]. CmWRKY42 can enhance SA accumulation by activating CmICS gene expression under red light, thereby increasing melon resistance to powdery mildew [36].
Seventy-five AtWRKY TFs were identified in Arabidopsis thaliana [37], 109 OsWRKYs in Oryza sativa [38], 85 SlWRKYs in Solanum lycopersicum [39], 61 CsWRKYs in Cucumis sativus L. [40], 63 ClWRKYs in Citrullus lanatus [41], and 57 CmWRKYs in Cucumis melo L. [42]. The molecular mechanisms of WRKY proteins in plants are mainly based on the following four aspects: (1) initiating plant responses to abiotic stress by directly regulating the expression of abiotic stress response-related genes, antioxidant signaling pathway genes, and their own genes; (2) orchestrating abiotic stress response networks by way of interacting with other proteins; (3) regulating signaling pathways, such as ABA signaling; (4) participating in plant physiological processes. In-depth insights into the regulatory mechanisms of WRKY TFs and related signaling pathways are expected to provide important theoretical contributions to plant biology [43].
The bottle gourd (Lagenaria siceraria), which originated in Africa more than 4000 years ago, belongs to the Cucurbitaceae family and is an important horticultural and medicinal crop [44]. The bottle gourd fruit is the main economic organ and can be used as a vegetable, container, or decoration. Young leaves and fruits are consumed as vegetables and contain a wide range of micro- and macronutrients and phytochemicals that are beneficial for human health [45]. Dried bottle gourds of various shapes can be made into specific containers for decoration or other uses at home. In addition, bottle gourds can be used as grafting stocks for cucumbers, melons, and watermelons to improve plant resistance [44]. Bottle gourds are cultivated worldwide, and there is a wide range of phenotypic variation in their agronomic traits, consistent with growers’ choices for desirable agronomic attributes and ethnobotanical utilization [46]. As bottle gourds exhibit great diversity in fruit shape, most studies have concentrated on the genetic structure of fruit shape variation [44]. With the completion of bottle gourd genome sequencing and the establishment of genetic transformation systems, a foundation has been established for further research on bottle gourd stress resistance [44,45]. In recent years, LsMLO genes have been identified, which play an important role in the response to powdery mildew stress in bottle gourds; however, there are few studies on the role of WRKY TFs in response to various stresses in bottle gourds [47].
In this study, we conducted a genome-wide characterization of LsWRKYs. We analyzed their sequence features, chromosomal distribution, evolutionary relationships, cis-regulatory elements, Gene Ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. To reveal the molecular mechanisms of bottle gourds in response to environmental stresses, gene expression profiling data were analyzed to determine the tissue specificity of LsWRKYs expression. Transcriptomic and qRT-PCR analyses provided information on the expression patterns of LsWRKYs in response to environmental stresses. This study contributes to the understanding of the LsWRKY gene family in responses to environmental stresses. It lays the basis for a comprehensive understanding of the functional mechanisms of environmental stress.

2. Materials and Methods

2.1. Identification of LsWRKYs in L. siceraria

L. siceraria genomic data were obtained from the CuGenDB website (http://cucurbitgenomics.org/organism/13, accessed on 13 August 2024). The hidden Markov model of WRKY structural domain (PF03106) was downloaded from the Pfam Protein Family Database (http://pfam.xfam.org/, accessed on 25 August 2024), which was used for searching WRKY-DNA-binding structural domains with an E-value threshold of ≤1e−15. To determine the reliability of HMMER results and the obtained LsWRKYs, the Conserved Domains Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 1 September 2024) and Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/, accessed on 5 September 2024) were employed. The physical and chemical properties of the LsWRKY proteins, such as the isoelectric point (pI) and molecular weight (MW), were predicted using the ExPASy proteomic server (http://web.expasy.org/protparam, accessed on 8 September 2024).

2.2. LsWRKYs Chromosomal Mapping, Gene Structure, and Motif Analysis

Chromosomal information for all identified LsWRKYs was sourced from the bottle gourd genome, and TBtools was used for chromosomal mapping of LsWRKYs [48]. The gene structure of LsWRKYs was predicted based on the genome sequence and coding sequences of each LsWRKY gene. The exon-intron structure of LsWRKYs was modeled using TBtools. The motifs within all LsWRKY protein sequences were analyzed using the MEME online program (http://meme.nbcr.net/meme/intro.html, accessed on 25 September 2024), and the maximum number of motifs selected was 10, with default parameters.

2.3. Multiple Sequence Alignment and Phylogenetic Analysis

All LsWRKY protein sequences were downloaded from the CuGenDB website (http://cucurbitgenomics.org/, accessed on 13 August 2024) and analyzed by comparison using ClustalW 2.0. Alignments of the LsWRKY amino acid sequences were produced using ClustalX with default settings. The LsWRKYs were classified into different groups. A total of 284 WRKY amino acid sequences of Arabidopsis thaliana, bottle gourd, cucumber, watermelon, and melon were used for the phylogenetic analysis. Neighbor-joining (NJ) phylogenetic trees were constructed using MEGA X software [40,41,42]. An evolutionary tree was visualized and optimized using the iTOL online tool (https://www.example.com, accessed on 9 October 2024).

2.4. Cis-Regulatory Elements in the Promoter of LsWRKYs

TBtools was utilized to obtain 2000 bp sequences of the upstream region in all LsWRKYs as a promoter. Cis-regulatory elements in the promoter region were analyzed using the online PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 October 2024). Cis-regulatory elements were visualized and optimized using the Gene Structure Display Server 2.0 tool (GSDS2.0, https://gsds.gao-lab.org/Gsds_help.php, accessed on 20 October 2024).

2.5. GO Term, KEGG Pathway, Collinearity, and Gene Duplication Analysis of LsWRKYs

The obtained LsWRKY proteins were analyzed for GO annotation using the Omicshare tool (https://www.omicshare.com/, accessed on 25 October 2024), and the obtained data were mapped and annotated [49]. The biological processes (BP), cellular components (CC), and molecular functions (MF) of the LsWRKY proteins were preliminarily analyzed. We used the eggNOG-mapper (http://eggnog-mapper.embl.de/, accessed on 30 October 2024) and Omicshare tools to perform the KEGG pathway enrichment analysis.
The Multiple Collinearity Scan toolkit (MCScanX) with default parameters was used to perform gene collinearity events between LsWRKY with WRKY genes from Arabidopsis thaliana, cucumber, watermelon, and melon. Collinearity analysis profiles of WRKY genes were visualized using TBtools v2.0 software. To investigate the selection pressure on LsWRKYs, the nonsynonymous substitution (Ka) and synonymous substitution (Ks) rates of duplicated genes were calculated using the KaKs_Calculator 2.0 [50].

2.6. Transcriptome Analysis of LsWRKYs in L. siceraria

The expression patterns of LsWRKY in L. siceraria var. USVL1VR-Ls were analyzed based on published RNA-seq data [51]. The transcripts-per-million values were calculated for five bottle gourd tissues: fruits, flowers, leaves, stems, and roots. Gene expression of the LsWRKYs was displayed in a heatmap produced using R v4.3 software, and the expression levels changed from blue to red, as shown by the color bar.
Using published RNA-seq datasets, LsWRKY expression was analyzed under heat, cold, and powdery mildew stress [47,52,53]. These transcriptome data were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 25 September 2024) using the SRA to Fastq plugin of TBtools software [48]. Low-quality transcriptomic data were filtered and de-joined using the Trimmomatic plugin. The reads were quantified directly as expression matrices using the Kallisto plugin and differentially expressed gene (DEG) analysis based on DESeq2. Transcriptome data were analyzed, and R software (with the package pheatmap) was used to display the expression of LsWRKY genes in a heatmap [54]. For transcriptome analysis of LsWRKYs in response to stresses, DEG was defined using a threshold of False discovery rate (or p-value) ≤ 0.05 and |log2 (fold change)| ≥ 1.5.

2.7. Plant Materials and Treatments

Bottle gourd (L. siceraria var. microcarpa) inbred line ‘Zhongyayaohulu’ was used in this study. Bottle gourd seeds were sown in a mixed substrate of peat, vermiculite, and perlite (1:1:1, v/v) and placed in a growth chamber with a 16/8 h diurnal cycle and 26/23 °C day/night temperature. The stress treatments were applied when the third true leaf appeared. And heat and cold treatment were performed at 45/40 °C and 6/4 °C day/night temperature with a 16/8 h diurnal cycle, respectively. Heart and cold treatment samples were collected at 0, 12, and 24 h and stored at −80 °C after snap-freezing using liquid nitrogen. At the three-leaf stage, the bottle gourd seedlings were treated with 400 mM NaCl. For the salt stress treatment, the second true leaves counted from the top buds were collected from seedlings at 0 and 5 d after treatment. Three biological replicates were used per treatment under the control and inoculation conditions described above.

2.8. RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis

Total RNA was extracted using a FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). First-strand cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). qRT-PCR primers were designed using DNAMAN v6.0 software (Table S1). The qRT-PCR method was described in our previous study [55]. The relative expression profile was determined using the 2−ΔΔCt method. Each sample was analyzed three times.

3. Results

3.1. Survey of LsWRKYs in Bottle Gourd

Fifty-one LsWRKYs were identified using HMMER (Table S2). Using the SMART and CDD databases for further confirmation, we demonstrated that these genes had single or double WRKY structural domains at the N-terminus, which is a defining feature of the WRKY gene family (Table S2). LsWRKY protein length ranged from 146 to 929 amino acids, with an average of 389. MWs ranged from 16.53 kDa (LsWRKY41) to 102.91 kDa (LsWRKY48), pIs from 4.64 (LsWRKY14) to 9.73 (LsWRKY36), and aliphatic indices from 31.81 (LsWRKY43) to 72.8 (LsWRKY45) (Table S3). WRKY proteins were generally cationic, unstable, and hydrophilic. The amino acid sequences of the LsWRKYs aligned with the defined AtWRKYs and were categorized into classes I, IIa, IIb, IIc, IId, IIe, and III (Figure 1). WRKY proteins contained two highly conserved structural domains: the WRKYGQK heptapeptide and the zinc-finger structural domain. In addition to WRKYGQK, other common heptapeptide residues were observed, including WRKYGKK (LsWRKY44) (Figure 1). Notably, a partial deletion of the zinc-finger sequence was observed in LsWRKY16/28/46 (Figure 1).
These genes were renamed LsWRKY1-LsWRKY51 based on their positional order on the chromosome (Figure 2). Chromosomes 3 and 5 contained the highest number of LsWRKY genes (eight). Chromosome 9 contained the lowest number of LsWRKY genes (one) (Figure 2). Chromosomes 8 and 10 contained two LsWRKY genes, chromosome 11 contained three LsWRKY genes, and four LsWRKY genes were present on chromosomes 1 and 6. Chromosome 2/4/7 contained relatively more LsWRKY genes (Figure 2). This suggested that although LsWRKY genes were present on all 11 chromosomes, their distribution on the respective chromosomes was heterogeneous.

3.2. Gene Structure and Conserved Motifs Analysis of LsWRKYs

To explore the diversity of LsWRKY gene functions, the exon-intron structure and conserved motifs of these genes were analyzed. The phylogenetic tree classification results were consistent with those described in Section 3.1, indicating that the LsWRKYs within the same class had comparable gene structure characteristics (Figure 3). Each LsWRKY gene consists of exons separated by a variable number of introns. Across classes, the number of introns (ranging from 3 to 17) and exons (ranging from 2 to 16) differed (Figure 3). Analyzing the structural diversity of exons and introns constituted a crucial element in the evolution of gene families and supported phylogenetic classification. These results suggest significant structural differences among LsWRKYs, which may correspond to functional diversity among closely related members. Ten motifs were analyzed using the online tool MEME, which varied in number within individual proteins (Figure 3 and Figure S1). All LsWRKY proteins contained a WRKY structural domain, and Motif 1 contained either the WRKYGQK or the WRKYGKK sequence (Figure 3). Motif 3 was present only in Class I, Motif 6 was specific to Class IId, and Motif 10 was specific to Class IIb (Figure 3). Motifs 7/8 were present in all members of Class IIa and b. Class III consisted only of motifs 1/2/4/9 (Figure 3). Most LsWRKY proteins in the same class had similar motifs and introns. These results suggest that motif analysis supports phylogenetic categorization, and different motif compositions may result in functional diversity of genes.

3.3. Phylogenetic Analysis of WRKY

To analyze the evolutionary relationship of LsWRKYs with WRKYs in other species, a phylogenetic tree was constructed using WRKY protein sequences from bottle gourd, Arabidopsis, cucumber, watermelon, and melon. The results showed that 284 WRKY proteins were divided into three distinct branches: Class I (n = 54), Class II (n = 191), and Class III (n = 39). Class II was further divided into Class IIa (n = 15), Class IIb (n = 26), Class IIc (n = 80), Class IId (n = 47), and Class IIe (n = 23), consistent with the results in Section 3.1 (Figure 4). The dispersed distribution of LsWRKYs in each class indicated that amplification of the WRKY family occurred prior to the differentiation of bottle gourds and Arabidopsis. In each evolutionary branch, there was a closer homology between bottle gourds and cucumber, watermelon, and melon than with Arabidopsis (Figure 4), which may reflect the functional diversification of the WRKY family during the evolution of various plants. In addition, the closer homology between bottle gourd and watermelon in comparison to that between cucumber and melon is consistent with the evolutionary outcome of Cucurbitaceae species.

3.4. Gene Duplication Analysis of LsWRKYs

The amplification mechanism of the LsWRKY family was investigated by collinearity and replicates analyses using BLASTP and MCScanX, respectively. Collinearity analysis of LsWRKYs revealed nine pairs of segmental duplications corresponding to 16 LsWRKYs (Figure 5 and Table S4). No tandem-duplicated genes were detected, suggesting that segmental duplication events were critical for the amplification of LsWRKYs during evolution. The phylogenetic mechanisms of the LsWRKY family were further investigated by building comparative collinearity maps of bottle gourds with four representative species: Arabidopsis thaliana, cucumber, watermelon, and melon. The WRKYs collinearity with Arabidopsis thaliana, cucumber, watermelon, and melon was 33, 49, 48, and 50, respectively (Figure 5 and Table S4). Bottle gourd, cucumber, watermelon, and melon belong to the Cucurbitaceae, and more than 94% of LsWRKYs were homozygous in cucumber, watermelon, and melon, suggesting that these WRKYs may have evolved from the same ancient WRKY gene (Figure 5 and Table S4). Thirty-three gene pairs were shared between bottle gourd and the other four species, suggesting that these gene pairs may have formed prior to the divergence of the different species (Figure 5 and Table S4). The Ka and Ks parameters were calculated to determine the selection pressure between segmental duplication events. Ka/Ks values ranged from 0.032 (LsWRKY31 vs. LsWRKY36) to 0.631 (LsWRKY45 vs. LsWRKY47) (Table S5). Notably, the Ka/Ks values were all <1, indicating that they were formed via purifying selection.

3.5. Cis–Regulatory Elements and GO Term and KEGG Pathways Analysis of LsWRKYs

To gain insights into the potential signaling pathways that may be involved in LsWRKYs, we used TBtools software to extract a 2 kb sequence in the LsWRKY upstream region, which was defined as the promoter sequence. Cis-regulatory elements involved in phytohormone regulation and stress response were investigated using the online tool PlantCARE, including five phytohormone-regulated cis-regulatory elements (ABA-responsive element, auxin-responsive element, gibberellin-responsive element, MeJA-responsive element, and SA-responsive element), and four stress-responsive cis-regulator elements (defense and stress-responsive element, drought-responsive element, low-temperature-responsive element, and wound-responsive element) (Figure 6 and Table S6). TC-rich repeats (defense- and stress-responsive elements) involved in defense and stress responses were also identified in the 17 LsWRKYs promoters, suggesting that these genes might be effective in the SA signaling pathway (Figure 6 and Table S6). The WUN-motif (a wound-responsive element) was found only in LsWRKY6/9. The cis-regulatory elements were specifically distributed in different phylogenetic clusters of the LsWRKY family. LTR (a low-temperature-responsive element) was detected in all classes, except Class IIa and Class IIb, indicating that Class IIa and Class IIb were insensitive to low temperatures (Figure 6 and Table S6). TC-rich repeats were found in all except Class IIc, and it was presumed that most classes were involved in defense and stress responses in bottle gourds. Light-responsive elements were detected in all LsWRKYs. A large proportion of these cis-regulatory elements were related to growth and stress responses, suggesting a potentially critical role for LsWRKYs in plant growth and development, as these cis-regulatory elements are widely and abundantly distributed in the promoters of almost all plants.
To obtain comprehensive insights into the function of LsWRKYs, we analyzed all LsWRKYs for GO term enrichment. A total of 75 GO terms were identified (Q-value ≤ 0.05), of which 51 belonged to BP, 9 belonged to CC, and 15 belonged to MF (Figure 7 and Table S7). In the BP class, most GO terms were associated with gene expression regulation and metabolism, such as the regulation of nucleic acid-templated transcription (Q-value = 3.56 × 10−48), cellular macromolecule metabolic process (Q-value = 3.48 × 10−17), and response to SA (Q-value = 0.038). In the CC class, most GO terms were closely associated with membranes, such as intracellular membrane-bound organelles (Q-value = 7.08 × 10−14) and membrane-bound organelles (Q-value = 7.08 × 10−14). In the MF class, all GO terms were related to binding, including transcription factor activity (Q-value = 1.36 × 10−62), sequence-specific DNA binding (Q-value = 2.91 × 10−69), and calmodulin binding (Q-value = 3.73 × 10−8) (Table S7). KEGG pathway enrichment analysis of LsWRKYs showed that two pathways were enriched, including ‘plant-pathogen interaction’ (Q-value = 2.06 × 10−13) and ‘MAPK signaling pathway’ (Q-value = 6.41 × 10−8) (Table S8). These results demonstrate that LsWRKYs are involved in bottle gourd growth and defense responses to environmental stress.

3.6. Transcriptome Analysis of LsWRKYs in Different Tissues Based on RNA-Sequencing Data

Tissue expression patterns of LsWRKYs were assessed based on bottle gourd available transcriptomic data, to elucidate the roles of LsWRKYs in plant growth and development. We used transcriptomic data to analyze the expression profiles of LsWRKYs in different organs, including roots, stems, leaves, flowers, and fruits. Notably, nine LsWRKYs (LsWRKY12/16/25/31/32/35/36/42/49) exhibited high expression in all tissues, whereas other LsWRKYs were significantly different in the roots, stems, leaves, flowers, and fruits (Figure 8). In the roots, LsWRKY8/12/16/32 were highly expressed and LsWRKY16/25/35/49 were highly expressed in the stems. LsWRKY2/8/16/35 were highly expressed in leaves, LsWRKY25/32/35/49 were highly expressed in flowers, and in fruits, LsWRKY8/16/25/35 showed high expression (Figure 8). These results suggest that LsWRKYs play a critical role in many aspects of bottle gourd growth and development.

3.7. Expression Pattern of LsWRKYs Under Abiotic and Biotic Stresses

WRKY TFs are specific to plants and well-known regulators of environmental stress-signaling pathways. To determine whether LsWRKYs were involved in abiotic and biotic stress responses, we used publicly available transcriptome information to generate gene expression profiles and obtained the combined expression patterns of LsWRKYs under heat, cold, and powdery mildew infection. The expression profiles showed that 11 LsWRKYs (LsWRKY14/16/18/19/21/24/33/34/38/39/44) and 14 LsWRKYs (LsWRKY2/9/14/16/18/19/21/34/38/39/43/44/47/51) were significantly altered under heat stress in the sensitive and tolerant varieties, respectively (Figure 9A). The transcriptional abundance of representative LsWRKYs in bottle gourd leaves under heat stress was detected by qRT-PCR within 24 h, which revealed different response patterns of LsWRKYs (Figure 9D). Throughout the study, the expression of LsWRKY2/9/14/18/34/43 showed an overall upward trend. The transcript abundance of LsWRKY34/47/53 were highest 12 h after heat stress, and LsWRKY34 were significantly upregulated and maintained a relatively high expression level 24 h after heat stress (Figure 9D). High-throughput sequencing results showed that the expression of LsWRKY14 was significantly reduced after cold stress, whereas the transcriptional abundance of LsWRKY2/5/8/9/12/13/18/19/21/25/25/30/32/36/37/38/39/40/43/44/47/48 was increased in treated leaves compared to that in the control (Figure 9C). qRT-PCR analysis showed that the expression of most LsWRKYs (except for LsWRKY14/48) showed an overall upward trend after cold stress (Figure 9E). Among them, the expression of LsWRKY2/8/9/12/18/38/43/44/47 were continuously upregulated after cold stress. After 12 h of cold stress, the transcriptional abundance of LsWRKY25/26/30/36/40 were the highest and remained at a relatively high expression level (Figure 9E). Under salt stress, numerous LsWRKYs exhibited differential expression. For bottle gourd seedlings treated with 120 h salt stress, the expressing of LsWRKY10/12/13/14/20/23/25/28/29/37/40/44 displayed significant upregulation, while dramatically decreased expression was observed for LsWRKY11/16/19/21/26/31/32/35/48/51 (Figure 9F). Based on RNA-seq data published by Wang et al., Transcriptome analysis identified LsWRKY genes as responsive to powdery mildew; four DEGs (LsWRKY9/18/19/21) and seven DEGs (LsWRKY2/8/18/19/21/32/43) were identified in resistant and susceptible varieties, respectively (Figure 9B) [47]. The transcript levels of LsWRKY18/19 were more strongly upregulated after powdery mildew infection compared to those in susceptible varieties (Figure 9B). LsWRKY9 was only upregulated in resistant bottle gourd after powdery mildew infection (Figure 9B). Notably, LsWRKY18 was enriched in the pathway of ‘plant-pathogen interaction’ and ‘MAPK signaling pathway’ (Table S8). The correlation analysis showed that LsWRKY9/18/19 were highly negatively correlated with MLO gene, and highly positively correlated with Disease resistant genes (Figure S2). LsWRKY9/18/19 may act as a positive regulator under powdery mildew infections. These findings support the hypothesis that, compared to sensitive varieties, LsWRKYs in tolerant bottle gourds have a higher acclimation state to environmental stress. LsWRKYs may play important roles in abiotic and biotic stress responses.

4. Discussion

WRKY TFs are a ubiquitous transcription factor class in the plant kingdom, which are regulated at the transcriptional, translational, genetic, and epigenetic levels, and are widely involved in a wide range of adversity stress responses in plants [8,9,10,14]. WRKY TFs have been identified in many plants, and studies have shown that CsWRKY1 and CsWRKY31 share 100% sequence similarity and jointly downregulate THCAS expression in cannabis [38,40,41,42,56]. L. siceraria is an economically important horticultural crop, and studying WRKY TFs in L. siceraria can help us understand their responses to environmental stress. This study identified 51 LsWRKYs in bottle gourds for the first time, and we analyzed their physicochemical properties, structural characteristics, evolutionary relationships, and expression patterns under stress. These findings provide valuable candidate TFs and lay a solid foundation for the development of new high-yield, multi-resistant varieties of bottle gourds.
WRKY TFs play key roles in molecular regulatory pathways, and the number of WRKY genes varies among plants during the evolution of species; for example, there are 63 WRKY genes in watermelon, 57 in melon, 61 in cucumber, 94 in sorghum, and more than 100 known WRKY members in rice [38,40,41,42]. The amplification or reduction of WRKY TFs is caused by environmental pressures and evolutionary forces, resulting in different evolutionary trajectories of WRKY genes within different species [57]. Fifty-one LsWRKYs were identified in bottle gourds, fewer than those identified in other Cucurbitaceae species. By comparing the number of WRKY TFs in Arabidopsis, tomato, rice, and cannabis, it was found that the reduction in the number of WRKY TFs in bottle gourds was mainly attributed to the lower number of Class III WRKY TFs (Figure 4). Similar findings have been reported for other cucurbit crops, such as cucumber, because there have been no recent repetitive events in their genomes [58]. Class III plays a key role in plant evolution and adaptation [59].
The most characteristic feature of WRKY TFs is that their protein sequences contain DNA-binding domains composed of segments of approximately 60 amino acids, with the highly conserved seven-amino-acid sequence WRKYGQK at the N terminus. The C-terminal end contains a conserved C2H2 (CX45CX22-23HX1H)-or C2HC (CX7CX23HXC)-type zinc-finger structure [21]. Most WRKY TFs have a conserved WRKYGQK motif; however, similar sequences were found in some WRKY TFs. This difference can severely affect the ability of WRKY proteins to bind W-box elements [60]. In soybean, two GmWRKY genes with the WRKYGKK motif did not bind to the W-box element [61]. NtWRKY12, containing WRKYGKK found in tobacco, recognized downstream binding sequences (TTTTCCAC) that were different from the W-box element [62]. WRKYGKK was found to be the most common mutation, which has been discovered in several plant species, including Arabidopsis, Oryza sativa L., Zea mays L., Vitis vinifera L., Solanum tuberosum L., and Malus pumila Mill. [21,63,64,65,66,67]. In the present study, only one variant of the WRKYGKK motif was found in Class IIc (LsWRKY44) (Figure 1). Modifications to the WRKYGQK motif may alter the DNA target binding specificity. Therefore, further investigations into the binding specificity and function of LsWRKY44 may be important. The iTAK database predicted 60 WRKY genes in bottle gourds, consistent with our HMMER-identified results (Table S2), of which nine genes (Lsi01G010800, Lsi01G011030, Lsi05G005230, Lsi05G020730, Lsi08G012440, Lsi08G012450, Lsi10G006720, Lsi10G015390, Lsi11G017190) were not defined as WRKY genes in this study. Sequence analysis revealed that these genes lack the highly conserved seven-amino acid sequence at the N terminus or the zinc-finger domain at the C terminus.
The Cucurbitaceae family is one of the most genetically diverse plant families worldwide. Genome sequencing has greatly promoted research on gene identification, genomic evolution, genetic variation, and molecular breeding of cucurbit crops. At least four other whole-genome duplication events occurred during the evolution of the Cucurbitaceae species belonging to the Benincaseae and Cucurbiteae form sister classes. The Benincaseae tribe is represented by four successively divergent genera: Cucumis, Benincasa, Lagenaria, and Citrullus. The history of speciation events indicates that the divergence time of bottle gourds and watermelons was shorter than that among other Benincaseae tribes. LsWRKYs were divided into three subgroups in bottle gourds: Class I, Class II, and Class III, distributed across 11 chromosomes, which is consistent with the results for cucumber, watermelon, and melon [40,41,42]. We found some LsWRKYs on the same branch as AtWRKY, CsWRKY, ClWRKY, and CmWRKY, suggesting that these WRKY TFs shared a common ancestor prior to differentiation. In addition, LsWRKY and ClWRKY had closer evolutionary relationships than other species, which might be related to the closer kinship between bottle gourds and watermelons, emphasizing the diversity and adaptability of WRKY TFs in different species [68].
Exon-intron structural analysis of WRKY TFs could reveal population-specific patterns, and LsWRKYs were in the same branch of the evolutionary tree and tended to have similar exon-intron structures. The number of introns in the LsWRKYs ranged from 3 to 17; this variation might be caused by gene duplication, inversion, and/or fusion events [69]. A conserved intron was present in the N-terminal WRKY domain of all 51 LsWRKYs, suggesting that this conserved intron may affect the alternative splicing of LsWRKY and its binding to downstream genes. The diversity of the LsWRKYs exon-intron structures could reflect the evolutionary diversity of the LsWRKY gene family. Analysis of the motif revealed structural conservation and diversity. Motif1 corresponded to the WRKY structural domain and was present in all LsWRKYs. Although the functions of most motifs are unknown, their distribution has a certain pattern that clearly shows the differences in the genes of different taxa. These motifs may indicate the involvement of WRKY TFs in specific biological processes and similar biological functions. WRKY proteins are widely distributed in plants and play key roles in important biological processes, including responses to abiotic (salt, heat, and cold stress) and biotic (downy mildew and powdery mildew infection) stresses [40,42]. Analysis of cis-regulatory elements in LsWRKYs may provide more information on LsWRKY gene expression. Many cis-regulatory elements, such as the TATA-box, CAAT-box, and A-box, are required for gene expression and are involved in the construction of transcription complexes [70]. In addition, there are cis-regulatory elements related to phytohormone regulation, such as ABA- and Auxin-responsive elements. Light-responsive cis-regulator elements, W-boxes, WUN-motifs, and TC repeats were also found in most LsWRKY promoters. There was also mutual regulation between the WRKY TFs [70]. Our analysis of cis-regulatory elements reflects the diversity of LsWRKYs expression regulation. This observation is consistent with the fact that bottle gourds exhibit strong environmental adaptability and are widely distributed worldwide.
In recent years, plant genome sequencing has revealed that 70–80% of angiosperms have undergone gene duplication events, including whole-genome and tandem duplication [71,72,73,74]. Tandem and segmental repeat events contribute to the amplification of WRKY genes [22]. Nine segmental duplication events were observed among the 16 LsWRKYs; however, no tandem duplication events were identified. These results are consistent with those on CsWRKY in cucumbers [40]. Segmental duplication was the main driver of WRKY TFs amplification and was one of the reasons why the number of WRKYs in cucurbits was lower than that in the other species.
Gene expression is closely associated with gene function [75]. In the present study, 51 LsWRKYs were analyzed for their expression in different tissues, including roots, stems, leaves, flowers, and fruits. LsWRKY8/16/12/32 was specifically expressed in roots, as previously reported, AtWRKY6/23/75 regulates Arabidopsis root development, and they have closely inherited homologs in bottle gourds [76,77]. Based on these results, genes specifically expressed in the roots could be recognized as key regulators of root development. They may play important roles in the response to various root stresses. Similarly, we also identified other possible key regulators in other tissues. WRKY proteins are widely involved in abiotic and biotic stress responses; at least 26 and 54 WRKY TFs have been identified to be involved in stress responses, respectively, in Arabidopsis and rice [78,79]. Transcript levels of CsWRKY9/18/48/57 were significantly altered in cucumber plants after heat or salt stress [40]. The homologous genes LsWRKY33/38/43/51 in bottle gourds also underwent significant changes after heat stress. Overexpression of AtWRKY30 may improve tolerance to oxidative and salt stress during seed germination [80]. The transcript levels of LsWRKY2/8/9/12/18/38/43/44/47 significantly rose continuously after cold stress, suggesting that these genes are important for regulating bottle gourd sensitivity to abiotic stress. 21 CsWRKYs were significantly altered by abiotic and biotic stresses, suggesting that some CsWRKYs have similar functions in response to abiotic and biotic stresses [40]. LsWRKYs in Class III are similar to WRKY TFs in other plants, responding to and defending against drought, salt, and cold stress through gene overexpression, silencing, or complementation [59]. OsWRKY45 and AtWRKY53 play important roles in response to drought and salt stress [81,82,83], and OsWRKY74 responds to cold environments [84]. qRT-PCR analysis showed that LsWRKY19/21/26/32, which belong to Class III, underwent significant changes under salt stress, whereas LsWRKY9/19/21/26/32/39 showed a strong induction in response to cold stress. Rice blasts activate OsWRKY67, and the overexpression of OsWRKY67 in rice enhances resistance to leaf blast, spike blight, and leaf blight [35]. In our study, the transcript levels of LsWRKY18/32/39/44 were upregulated by powdery mildew infection, and LsWRKY8 was only upregulated in the resistant varieties, suggesting that LsWRKYs might play an important regulatory role in biotic resistance. In addition, WRKY TFs are known to function in conjunction with a variety of signaling cascades, including SA, JA, MAPK, Ca2+, and ROS [57], suggesting that the specific function of LsWRKYs requires further functional validation and signaling crosstalk examination. These results provide a basis for further studies on the function of LsWRKYs and genetic improvement of bottle gourd resistance to biotic and abiotic stresses.

5. Conclusions

Fifty-one LsWRKYs were identified in bottle gourds. Basic information, chromosomal distribution, phylogenetics, gene and motif analysis, GO terms, and cis-regulator element analysis were produced. Analysis of gene expression patterns suggested that LsWRKYs are specifically expressed in different plant tissues, and some LsWRKYs may play important roles in plant stress tolerance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101192/s1, Figure S1: The identified motifs in LsWRKY proteins are indicated and named Motifs 1-10; Figure S2: Correlation analysis between LsWRKYs and resistance genes; Table S1: List of quantitative real-time PCR (qRT-PCR) primers for expression analysis of LsWRKY genes; Table S2: Identification of conserved domains in LsWRKY proteins; Table S3: Characteristics of the identified LsWRKY genes in Lagenaria siceraria; Table S4: Collinearity analysis of WRKY genes among bottle gourd with Arabidopsis thaliana, cucumber, watermelon and melon; Table S5: Estimated Ka/Ks rates of duplicated WRKY genes in L. siceraria; Table S6: Analysis of cis-regulator elements in the promoter regions of LsWRKYs; Table S7: Complete list of overrepresented gene ontology (GO) terms for LsWRKYs; Table S8: Complete list of enriched KEGG pathways for LsWRKYs.

Author Contributions

Conceptualization, H.J. and S.W.; methodology, S.W. and H.J.; software, W.L. and S.T.; investigation, S.W. and W.L.; writing—original draft preparation, S.W.; writing—review and editing, H.J.; visualization, H.J., S.W. and W.L.; resources, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (ZR2023QC248), Liaocheng University, China (grant numbers 318052244, 318052290, and 31946221226), the Key Research and Development Program of Liaocheng (grant number 2022YDNY11), and Liaocheng “Science and Technology Vice President” Collaborative Innovation Project (grant number 2024XT07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Alignment of 51 LsWRKY domain sequences in bottle gourd. For Class I WRKY proteins, N-terminal and C-terminal WRKY domains are represented by ‘N’ and ‘C’, respectively. The typical amino acid residues within the WRKY domain and zinc-finger motif are in black and red, respectively.
Figure 1. Alignment of 51 LsWRKY domain sequences in bottle gourd. For Class I WRKY proteins, N-terminal and C-terminal WRKY domains are represented by ‘N’ and ‘C’, respectively. The typical amino acid residues within the WRKY domain and zinc-finger motif are in black and red, respectively.
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Figure 2. Chromosomal locations of LsWRKYs in bottle gourd. The scale bar of chromosomes is shown on the left.
Figure 2. Chromosomal locations of LsWRKYs in bottle gourd. The scale bar of chromosomes is shown on the left.
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Figure 3. Conserved motifs and gene structure diagram of LsWRKYs. Evolutionary relationship, exon-intron structures and conserved motifs of LsWRKYs. Left panel: the phylogenetic tree was constructed from the WRKY domain sequences of LsWRKY proteins. The various groups and subgroups are shown in different colors. Middle panel: gene structure of LsWRKY TFs. Untranslated 5’- and 3’-regions, exons, and introns are indicated by green boxes, yellow boxes, and black lines, respectively. Right panel: the motifs are represented by different colored boxes. Details of each motif are shown in Figure S1.
Figure 3. Conserved motifs and gene structure diagram of LsWRKYs. Evolutionary relationship, exon-intron structures and conserved motifs of LsWRKYs. Left panel: the phylogenetic tree was constructed from the WRKY domain sequences of LsWRKY proteins. The various groups and subgroups are shown in different colors. Middle panel: gene structure of LsWRKY TFs. Untranslated 5’- and 3’-regions, exons, and introns are indicated by green boxes, yellow boxes, and black lines, respectively. Right panel: the motifs are represented by different colored boxes. Details of each motif are shown in Figure S1.
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Figure 4. Phylogenetic tree of the total WRKY proteins from bottle gourd, Arabidopsis, cucumber, watermelon, and melon. The phylogenetic tree was constructed using the neighbor-joining method with a bootstrap test of 1000 times based on MEGA X software. WRKY TFs were categorized into seven subgroups, including Class I, Class IIa–e, and Class III (shown in different colors).
Figure 4. Phylogenetic tree of the total WRKY proteins from bottle gourd, Arabidopsis, cucumber, watermelon, and melon. The phylogenetic tree was constructed using the neighbor-joining method with a bootstrap test of 1000 times based on MEGA X software. WRKY TFs were categorized into seven subgroups, including Class I, Class IIa–e, and Class III (shown in different colors).
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Figure 5. Schematic representations for the interchromosomal relationships of WRKYs of bottle gourd with those of Arabidopsis thaliana, cucumber, watermelon, and melon. Collinearity was visualized using the Collinearity Plot in TBtools. (A) Collinearity analysis of WRKYs between bottle gourd and Arabidopsis thaliana. (B) Collinearity analysis of WRKYs between bottle gourd and cucumber. (C) Collinearity analysis of WRKYs between bottle gourd and watermelon. (D) Collinearity analysis of WRKYs between bottle gourd and melon. Boxes represent chromosomes, and lines represent segmental duplication gene pairs.
Figure 5. Schematic representations for the interchromosomal relationships of WRKYs of bottle gourd with those of Arabidopsis thaliana, cucumber, watermelon, and melon. Collinearity was visualized using the Collinearity Plot in TBtools. (A) Collinearity analysis of WRKYs between bottle gourd and Arabidopsis thaliana. (B) Collinearity analysis of WRKYs between bottle gourd and cucumber. (C) Collinearity analysis of WRKYs between bottle gourd and watermelon. (D) Collinearity analysis of WRKYs between bottle gourd and melon. Boxes represent chromosomes, and lines represent segmental duplication gene pairs.
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Figure 6. Distribution of cis-regulatory elements in promoters of LsWRKYs. Differently colored boxes represent ABA-responsive element, auxin-responsive element, defense- and stress-responsive element, drought-responsive element, gibberellin-responsive element, light-responsive element, low-temperature-responsive element, MeJA-responsive element, SA-responsive element, and wound-responsive element, respectively.
Figure 6. Distribution of cis-regulatory elements in promoters of LsWRKYs. Differently colored boxes represent ABA-responsive element, auxin-responsive element, defense- and stress-responsive element, drought-responsive element, gibberellin-responsive element, light-responsive element, low-temperature-responsive element, MeJA-responsive element, SA-responsive element, and wound-responsive element, respectively.
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Figure 7. GO analysis of identified LsWRKYs in Biological Process (BP). GO terms with a Q-value ≤ 0.05 were considered enriched. The color and size of filled circles indicate the Q-value and LsWRKYs number, respectively, in enriched terms.
Figure 7. GO analysis of identified LsWRKYs in Biological Process (BP). GO terms with a Q-value ≤ 0.05 were considered enriched. The color and size of filled circles indicate the Q-value and LsWRKYs number, respectively, in enriched terms.
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Figure 8. Expression profiles of LsWRKYs in L. siceraria var. USVL1VR-Ls diverse tissues determined based on RNA-seq [51]. (A) Heatmap of LsWRKYs in flower, stems, fruit, leaf, and root of bottle gourd. (B) The top 4 LsWRKYs in terms of expression levels are presented for each tissue.
Figure 8. Expression profiles of LsWRKYs in L. siceraria var. USVL1VR-Ls diverse tissues determined based on RNA-seq [51]. (A) Heatmap of LsWRKYs in flower, stems, fruit, leaf, and root of bottle gourd. (B) The top 4 LsWRKYs in terms of expression levels are presented for each tissue.
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Figure 9. Expression analysis of LsWRKY genes under different environmental stressors. Heat map of LsWRKYs expression in bottle gourd under normal growth conditions (Control) and two different environmental stresses: heat stress (A) and powdery mildew (B). The color scale at the top of each heatmap represents the transcripts-per-million expression values. Red indicates higher levels, and blue indicates lower levels. (C) The volcano plot shows LsWRKYs that changed significantly under cold stress conditions. Red and blue dots indicate genes that increased and decreased significantly, respectively. Gray dots indicate genes that did not change significantly. Control represents the untreated sample. Expression profiles of representative LsWRKYs in bottle gourd after heat stress (D), cold stress (E), and salt stress (F). Different letters and asterisk indicate significant differences in the expression of representative LsWRKYs between the control and other time points (one-way analysis of variance test, p < 0.05).
Figure 9. Expression analysis of LsWRKY genes under different environmental stressors. Heat map of LsWRKYs expression in bottle gourd under normal growth conditions (Control) and two different environmental stresses: heat stress (A) and powdery mildew (B). The color scale at the top of each heatmap represents the transcripts-per-million expression values. Red indicates higher levels, and blue indicates lower levels. (C) The volcano plot shows LsWRKYs that changed significantly under cold stress conditions. Red and blue dots indicate genes that increased and decreased significantly, respectively. Gray dots indicate genes that did not change significantly. Control represents the untreated sample. Expression profiles of representative LsWRKYs in bottle gourd after heat stress (D), cold stress (E), and salt stress (F). Different letters and asterisk indicate significant differences in the expression of representative LsWRKYs between the control and other time points (one-way analysis of variance test, p < 0.05).
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Jin, H.; Wang, S.; Li, W.; Tan, S.; Zhao, Y. Comparative Identification of LsWRKY Transcription Factors and Transcriptional Response to Abiotic and Biotic Stresses in Lagenaria siceraria. Horticulturae 2025, 11, 1192. https://doi.org/10.3390/horticulturae11101192

AMA Style

Jin H, Wang S, Li W, Tan S, Zhao Y. Comparative Identification of LsWRKY Transcription Factors and Transcriptional Response to Abiotic and Biotic Stresses in Lagenaria siceraria. Horticulturae. 2025; 11(10):1192. https://doi.org/10.3390/horticulturae11101192

Chicago/Turabian Style

Jin, Han, Shuoshuo Wang, Wenli Li, Shujing Tan, and Yan Zhao. 2025. "Comparative Identification of LsWRKY Transcription Factors and Transcriptional Response to Abiotic and Biotic Stresses in Lagenaria siceraria" Horticulturae 11, no. 10: 1192. https://doi.org/10.3390/horticulturae11101192

APA Style

Jin, H., Wang, S., Li, W., Tan, S., & Zhao, Y. (2025). Comparative Identification of LsWRKY Transcription Factors and Transcriptional Response to Abiotic and Biotic Stresses in Lagenaria siceraria. Horticulturae, 11(10), 1192. https://doi.org/10.3390/horticulturae11101192

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