Uncovering the Genetic Regulation Associated with Etofenprox-Induced Phytotoxicity in Soybean

다국어 초록 (Multilingual Abstract)

Etofenprox, a synthetic pyrethroid insecticide widely used in soybean cultivation, causes distinct phytotoxicity in certain cultivars. Reports of pesticide-induced crop damage exist for several species; however, studies on differential phytotoxic responses among soybean (Glycine max (L.) Merrill) cultivars, particularly to etofenprox, have only recently begun to emerge. Consequently, elucidating the mechanisms underlying etofenprox-induced phytotoxicity and identifying the causal genes is essential not only for stable soybean production and the breeding of tolerant cultivars, but also for advancing our understanding of pesticide-induced phytotoxicity mechanisms across diverse crops. This study aimed to validate key genes involved in the etofenprox-induced phytotoxicity response and to clarify the underlying mechanisms by analyzing gene expression. To compare expression patterns following etofenprox treatment, real-time quantitative reverse transcription PCR (qRT-PCR) was performed for the candidate genes Glyma.16G182200 (GPI-anchored adhesin-like protein) and Glyma.16G182300 (CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR SUBUNIT 100, CPSF100), which were identified in previous research. The qRT-PCR results revealed distinct expression patterns between the two cultivars. In the sensitive cultivar ‘Danbaek’, Glyma.16G182200 was significantly downregulated, whereas Glyma.16G182300 was upregulated, peaking at 24 hours post-treatment. In contrast, the insensitive cultivar ‘Williams82’ showed no significant changes in the expression of either gene, suggesting that the altered expression of these genes is specific to the phytotoxicity response. RNA-seq analysis of ‘Danbaek’ revealed distinct temporal gene expression patterns in response to phytotoxicity. From the RNA-seq analysis, a total of 1,361 DEGs were identified between the control and etofenprox-treated samples at 12 h, and 951 genes were significant at 24 h post treatment. At 12 h post-treatment, upregulated differentially expressed genes (DEGs) were significantly enriched in DNA repair and photosystem stabilization, whereas genes related to translation and oxidative phosphorylation were downregulated. By 24 h, the molecular response shifted towards active defense mechanisms, characterized by the upregulation of salicylic acid biosynthetic processes and iron ion transport. Notably, KEGG pathway analysis highlighted the consistent activation of MAPK signaling and plant-pathogen interaction pathways, while photosynthesis and energy metabolism were suppressed throughout the treatment. These results suggest that ‘Danbaek’ recognizes etofenprox as a pathogen-like stressor and activates similar defense mechanisms. This response prioritizes stress signaling and pathogen defense responses over growth and energy production. Notably, significant expression changes were observed among transcription factors, prompting further investigation into the regulatory mechanisms of the candidate genes. To investigate the regulatory mechanisms underlying the differential expression patterns of the candidate genes, promoter motif analysis was performed on Glyma.16G182200 and Glyma.16G182300. Promoter sequences were analyzed across 10 soybean cultivars to identify cis-regulatory motifs that may contribute to cultivar-specific differences in transcriptional regulation. For Glyma.16G182200, 21 known motifs were identified in ‘Williams82’, while 20 motifs were conserved in ‘Danbaek’. In ‘Danbaek’ W-box motif was uniquely disrupted by a specific single nucleotide polymorphism (A→G) at position 34,304,640 in the promoter region. Analysis of Glyma.16G182300 revealed 20 motifs in ‘Williams82’, with the MBSI (MYB Binding Site I) motif absent in ‘Danbaek’. This absence is attributed to a deletion at positions 34,307,008–34,307,009, located approximately 300 bp upstream of the transcription start site. These structural variations in promoter regions were consistently observed in the sensitive cultivars ‘Danbaek’ and ‘‘Kwangan’’, while insensitive cultivars retained intact motif sequences. Consequently, the absence of W-box and MBSI motifs may contribute to altered transcriptional regulation and cultivar-specific sensitivity to etofenprox. Overall, these findings suggest that the phytotoxicity observed in the sensitive cultivar ‘Danbaek’ is associated with excessive activation of immune signaling pathways, likely driven by promoter variations disrupting key transcription factor binding sites. This study provides insights into the molecular mechanisms underlying cultivar-specific susceptibility to etofenprox.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• 1.1 Research background and necessity 1
• 1.2 Research objectives and content 3
• Ⅱ. LITERATURE REVIEW 5
• 2.1 Phytotoxicity and plant defense mechanisms 5
• 2.2 Etofenprox-induced phytotoxicity in soybean 8
• Ⅲ. MATERIALS AND METHODS 11
• 3.1 Plant materials and growth conditions 11
• 3.2 Quantitative reverse transcription PCR (qRT-PCR) 12
• 3.2.1 RNA extraction and primer design 12
• 3.2.2 qRT-PCR conditions and data analysis 15
• 3.3 RNA sequencing (RNA-seq) analysis 15
• 3.3.1 Data processing and identification of DEGs 15
• 3.3.2 Gene Ontology (GO) and KEGG pathway enrichment analysis 18
• 3.4 Cis-regulatory motif identification in candidate gene promoters 19
• Ⅳ. RESULTS 20
• 4.1 Expression validation of candidate genes by qRT-PCR 20
• 4.2 Transcriptome-wide response to etofenprox treatment 23
• 4.2.1 Data processing and identification of DEGs 23
• 4.2.2 Gene Ontology (GO) and KEGG pathway enrichment analysis 32
• 4.2.3 Expression profiles of key signaling and defense-related genes 36
• 4.3 Cis-regulatory element analysis in candidate gene promoters 47
• Ⅴ. DISCUSSION 54
• 5.1 Identification of key genes associated with etofenprox sensitivity 54
• 5.2 Functional implications of etofenprox-responsive metabolic pathways 56
• 5.2.1 Upregulated at 12 hpt: Developmental transition and DNA maintenance 56
• 5.2.2 Downregulated at 12 hpt: Growth suppression and metabolic slowdown · 60
• 5.2.3 Upregulated at 24 hpt: Defense activation and specialized metabolism 65
• 5.2.4 Downregulated at 24 hpt: Iron homeostasis disruption and photosynthetic decline 67
• 5.2.5 Commonly regulated genes: Sustained defense responses 69
• 5.2.6 Integrative analysis: Progressive metabolic dysfunction model 73
• 5.3 Transcriptional regulation mediated by promoter motif variations 74
• 5.4 Integrative model of etofenprox resistance mechanism in soybean 78
• 5.4.1 Structural vulnerability and mechanotransduction 78
• 5.4.2 Self-destructive signal amplification 80
• 5.4.3 Dysregulated RNA processing and immune homeostasis 81
• Ⅵ. CONCLUSION 83
• REFERENCES 89
• ABSTRACT 97