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<p>To explore the molecular mechanisms by which exogenous 2,4-epibrassinolide (EBR) alleviates the cadmium stress on soybean (<i>Glycine max</i> (L.) Merr.), we performed methylation-sensitive amplified polymorphism (MSAP) detection and methylation differential gene analysis on soybean seedlings after 3-day cadmium stress. We also conducted transcriptome sequencing (RNA-seq) and quantitative fluorescence validation of selected genes. We divided the soybean seedlings used for experiments into 3 groups: untreated control (CK), cadmium stress (Cd), and EBR-cadmium stress (Cd-EBR) groups. The results showed that EBR reduced the DNA methylation level in the leaves of cadmium-tolerant soybean seedlings. With the increasing cadmium stress concentrations, the ability of EBR to reduce DNA methylation level decreased, and non-methylation (type I) was the predominant DNA methylation type under all treatments. We found that some regulatory elements play an important role in stress tolerance. RNA-seq results demonstrated that the number of differentially expressed genes (DEGs) in the comparison of CK vs. Cd was the largest, and the number of DEGs in the comparison of CK vs. Cd-EBR was the smallest, and in each pairwise comparison, the number of down-regulated genes was higher than that of up-regulated genes. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly annotated to GO terms such as cell, cell part, organelle, membrane, membrane part, catalytic activity, binding, cellular process, metabolic process, and single-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that DEGs in Cd vs. Cd-EBR were significantly enriched in pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, stilbenoid, diarylheptanoid and gingerol biosynthesis, and plant-pathogen interaction, suggesting that these genes might indicate that EBR directly or indirectly participates in the growth and development of soybean seedlings under EBR and cadmium stresses, and EBR alleviated Cd stress on soybean seedlings by regulating the expression of these genes. Overall, this study reveals the molecular mechanisms by which EBR alleviates Cd stress on soybean seedlings. Our findings provide valuable references for improving soybean growth under heavy metal Cd stress by application of EBR.</p>

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MSAP-Based Assay and Transcriptomics Analysis Reveal Molecular Mechanisms by Which Exogenous 2,4- Epibrassinolide Alleviates Cadmium Stress on Soybean Seedlings

  • 期刊论文

  • 发表日期：

    2026年4月13日

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  • Yanjie Wang,
  • Xiaoping Liang,
  • Yujing Gao,
  • Zhihui Hu

摘要

To explore the molecular mechanisms by which exogenous 2,4-epibrassinolide (EBR) alleviates the cadmium stress on soybean (Glycine max (L.) Merr.), we performed methylation-sensitive amplified polymorphism (MSAP) detection and methylation differential gene analysis on soybean seedlings after 3-day cadmium stress. We also conducted transcriptome sequencing (RNA-seq) and quantitative fluorescence validation of selected genes. We divided the soybean seedlings used for experiments into 3 groups: untreated control (CK), cadmium stress (Cd), and EBR-cadmium stress (Cd-EBR) groups. The results showed that EBR reduced the DNA methylation level in the leaves of cadmium-tolerant soybean seedlings. With the increasing cadmium stress concentrations, the ability of EBR to reduce DNA methylation level decreased, and non-methylation (type I) was the predominant DNA methylation type under all treatments. We found that some regulatory elements play an important role in stress tolerance. RNA-seq results demonstrated that the number of differentially expressed genes (DEGs) in the comparison of CK vs. Cd was the largest, and the number of DEGs in the comparison of CK vs. Cd-EBR was the smallest, and in each pairwise comparison, the number of down-regulated genes was higher than that of up-regulated genes. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly annotated to GO terms such as cell, cell part, organelle, membrane, membrane part, catalytic activity, binding, cellular process, metabolic process, and single-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that DEGs in Cd vs. Cd-EBR were significantly enriched in pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, stilbenoid, diarylheptanoid and gingerol biosynthesis, and plant-pathogen interaction, suggesting that these genes might indicate that EBR directly or indirectly participates in the growth and development of soybean seedlings under EBR and cadmium stresses, and EBR alleviated Cd stress on soybean seedlings by regulating the expression of these genes. Overall, this study reveals the molecular mechanisms by which EBR alleviates Cd stress on soybean seedlings. Our findings provide valuable references for improving soybean growth under heavy metal Cd stress by application of EBR.