Multi-omics integration identifies GmISO5 as a key regulator of seed isoflavone biosynthesis and implicates it in the response to soybean mosaic virus.

INTRODUCTION Isoflavones are specialized metabolites that accumulate abundantly in soybean seeds, contributing to nutritional quality, human health benefits, and plant defense. Although the core isoflavone biosynthetic pathway has been well characterized, its upstream regulatory network and potential relationship with plant stress responses remain incompletely understood.

Objectives

This study aimed to identify key regulators of seed isoflavone accumulation and to assess whether the identified regulator is associated with the soybean response to soybean mosaic virus (SMV) infection.

Methods

We integrated genome-wide and transcriptome-wide association analyses with CRISPR/Cas9-mediated knockout, transcriptome profiling, molecular interaction assays, co-expression network analysis, and population genetic analysis.

Results

GmISO5 was identified as a major genetic determinant of seed isoflavone content. CRISPR/Cas9-mediated knockout of GmISO5 reduced total seed isoflavones by approximately 80% and broadly altered the expression of isoflavone-pathway genes. Molecular assays demonstrated that GmISO5 directly binds to and activates the promoters of GmHIDH and GmCHS1, which encode 2-hydroxyisoflavanone dehydratase and chalcone synthase, respectively, demonstrating direct regulation at distinct steps of the isoflavone biosynthetic pathway. Transcriptome profiling and co-expression network analysis further placed GmISO5 within an isoflavone-enriched regulatory module. Following SMV inoculation, GmISO5 knockout lines exhibited more severe symptoms and higher viral accumulation, implicating GmISO5 in the response to SMV infection. Population genetic analyses showed that the low-isoflavone haplotype (GmISO5Low) reached 91% frequency in improved cultivars, suggesting that its enrichment may have resulted from indirect selection for seed-weight and oil-content traits associated with GmISO5Low rather than direct selection for reduced isoflavone accumulation.

Conclusion

GmISO5 is a key regulator of soybean seed isoflavone accumulation that directly activates GmHIDH and GmCHS1. The increased SMV susceptibility of GmISO5 knockout lines further implicates GmISO5 in the soybean response to SMV infection, although whether reduced isoflavone accumulation directly causes the altered SMV phenotype remains to be established.