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. 2025 May 9;25(1):607.
doi: 10.1186/s12870-025-06606-7.

Transcriptome analysis and genome-wide identification of WRKY gene family in Leonurus japonicus under drought stress

Tongtong Guo #  1 , Qing Wang #  1 , Yuxiao Yi  1 , Zelin Yin  1 , Lili Xu  1 , Fengtang Yang  2 , Jianing Xu  3 , Ming Zhang  4
Affiliations

Transcriptome analysis and genome-wide identification of WRKY gene family in Leonurus japonicus under drought stress

Tongtong Guo et al. BMC Plant Biol. .

Abstract

Background: Leonurus japonicus (L. japonicus) is a herbaceous flowering plant, widely distributed in Asia. Drought is one of the primary environmental stress factors affecting L. japonicus growth. Previous studies have demonstrated that WRKY transcription factors (TFs) play a crucial role in plant responses to drought stress. So far, there has been no research on the function of WRKY genes in L. japonicus.

Results: The physiological experiment results showed that drought stress significantly increased the malondialdehyde (MDA), proline, and hydrogen peroxide (H₂O₂) content of L. japonicus. Transcriptome analysis revealed significant changes in the expression levels of the WRKY gene family. Based on bioinformatics analysis, 67 WRKY genes (LjWRKYs) were identified in the genome of L. japonicus, with amino acid lengths ranging from 85 to 574. The LjWRKYs can be divided into three subfamilies. Among them, the expression of LjWRKY (1/4/23/44) were significantly up-regulated under drought stress, whereas the expression of LjWRKY (21/25/65) were significantly down-regulated. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that after drought stress, differentially expressed genes (DEGs) were enriched in plant hormone signal transduction pathway, the MAPK signaling pathway and biosynthesis of secondary metabolites pathway. In the MAPK pathway, there were 19 DEGs, 9 of which contained W-box regions, suggesting that they may be potential regulatory targets of LjWRKY TFs under drought stress.

Conclusion: These findings suggested that WRKY gene family may participate in the response to drought stress in L. japonicus. This study provides a scientific basis for the further development and functional validation of the WRKY gene family in L. japonicus.

Clinical trial number: Not applicable.

Keywords: Leonurus japonicus; Drought stress; Genome-wide analysis; WRKY family.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: Plant material used in the study complies with relevant institutional, national, and international guidelines and legislation. Plant material used in the study is not wild species and also permission for the plant material was obtained. All experiments were performed according to institutional guidelines of Shandong University of technology, China. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Physiological responses of L. japonicus to simulated drought stress induced by PEG6000 treatment. (A) Dynamic effects of different concentrations of PEG 6000 treatment on the growth of L. japonicus at 0, 4, 8, and 12 h. (B) MDA content under simulated drought stress. (C) Proline content under simulated drought stress. (D) H2O2 content under simulated drought stress. Asterisks indicate significant differences with respect to values of 0 h (Student’s t-test): **, P < 0.01
Fig. 2
Fig. 2
Transcriptome analysis of L. japonicus under simulated drought stress. (A) PCA analysis. (B) Differentially Expressed Genes (DEGs) under simulated drought stress. UP, up-regulated DEGs, DOWN, down-regulated DEGs. (C) Top 20 bubble plot of KEGG enrichment analysis for DEGs. (D) Number of the top 20 transcription factor families identified in the transcriptome analysis of L. japonicus under simulated drought stress
Fig. 3
Fig. 3
Chromosomal localization of LjWRKYs in L. japonicus
Fig. 4
Fig. 4
Intron-Exon structure and motifs in LjWRKYs of L. japonicus. Schematic representation of the WRKY genes, including 3’ and 5’ UTR (untranslated regions) and CDS (coding sequences)
Fig. 5
Fig. 5
Phylogenetic tree of WRKY proteins from L. japonicus and A. thaliana
Fig. 6
Fig. 6
Multiple sequence alignment of the WRKY domains of 67 LjWRKY proteins. The red box represents the WRKY domain, and the blue box represents the zinc finger structure
Fig. 7
Fig. 7
Syntenic analysis of LjWRKYs in L. japonicus. (A) Circos plot showing the gene duplication events of LjWRKYs within the L. japonicus genome. The red lines indicate duplicated gene pairs. (B) Syntenic relationships of WRKY genes among L. japonicus, A. thaliana, and S. bowleyana. Blue lines represent syntenic WRKY gene pairs between the species
Fig. 8
Fig. 8
Cis-acting elements in the promoter regions of LjWRKYs in L. japonicus
Fig. 9
Fig. 9
Expression patterns of the LjWRKYs in different tissues and under drought stress. (A) The expression patterns of LjWRKYs in different tissues, including leaf, stem, flower, and root. The color gradient ranges from red (high expression) to white (low expression), indicating variations in expression levels. (B) The expression changes of LjWRKYs under drought stress treatments, represented by three biological replicates (PEG-1, PEG-2, PEG-3), are shown as log₂ fold change (log₂FC) values relative to the control group (CK). The color gradient ranges from purple (high expression) to cyan (low expression), illustrating the magnitude of expression changes. Gene grouping is based on the phylogenetic tree classification Groups I to III
Fig. 10
Fig. 10
Expression heatmap of MAPK pathway genes in L. japonicus under drought stress. The expression changes of LjWRKYs under drought stress treatments, represented by three biological replicates (PEG-1, PEG-2, PEG-3), are shown as log₂ fold change (log₂FC) values relative to the CK. Purple indicates up-regulation, and blue indicates down-regulation. The genes marked with a five-pointed star contain W-box elements
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