Multi‐Omics Integration Reveals Heavy Ion‐Induced Enhancement of Soybean Isoflavone Biosynthesis

Isoflavones are valuable bioactive compounds in soybeans with significant therapeutic potential, yet conventional breeding approaches face limitations in enhancing their accumulation. We hypothesized that heavy ion radiation could effectively stimulate isoflavone biosynthesis through coordinated molecular reprogramming mechanisms. To test this hypothesis, we conducted an integrated transcriptomic-proteomic analysis investigating radiation effects on isoflavone metabolism across four developmental stages in soybean. Heavy ion treatment validation confirmed our hypothesis, substantially increasing isoflavone accumulation with total isoflavones showing significant enhancement (p = 7.34 × 10^-6), alongside specific increases in genistin (p = 3.46 × 10^-4) and genistein (p = 1.61 × 10^-4). Multi-omics profiling revealed the molecular basis underlying these metabolic changes: RNA sequencing identified 3639 differentially expressed genes, while quantitative proteomics revealed 1458 differentially expressed proteins, indicating extensive macromolecular reprogramming in response to radiation treatment. Integration of transcriptomic and proteomic datasets revealed coordinated regulatory networks driving enhanced isoflavone production. Pathway enrichment analysis identified 89 overlapping KEGG pathways, with 33 showing significant co-enrichment (p < 0.05). Six key pathways exhibited coordinated upregulation: pentose phosphate pathway, glutathione metabolism, amino acid biosynthesis, lipid metabolism, flavonoid biosynthesis, and fatty acid synthesis. Notably, glutathione metabolism was most extensively regulated (12 genes, 27 proteins), suggesting that enhanced isoflavone production functions as part of an integrated antioxidant defense mechanism triggered by radiation stress. The tight coordination between molecular and metabolic responses was demonstrated through strong correlations (r > 0.8, p < 0.01) between mRNA expression, protein abundance, and metabolite accumulation. RT-qPCR validation confirmed transcriptomic findings (r > 0.85, p < 0.001), supporting the reliability of our multi-omics approach. These results establish heavy ion radiation as an effective biotechnological tool for enhancing secondary metabolite production and provide mechanistic insights into coordinated macromolecular responses that could inform future crop improvement strategies.