Genome-wide transcriptome analysis reveals transcription factors associated with isoflavone content in soybean

Isoflavones are pivotal secondary metabolites primarily found in leguminous plants. Soybean isoflavones not only offer multiple health benefits for humans but also play crucial roles in shaping plant disease resistance and soybean-rhizobia interactions. However, the regulatory mechanisms underlying soybean isoflavone biosynthesis remain largely unknown. In this study, the quantification of seed isoflavones in extreme phenotype materials revealed that isoflavone accumulation mainly occurred during late seed development. Transcriptomic profiling demonstrated significant enrichment of differentially expressed genes (DEGs) in the isoflavone biosynthetic pathway at 60 days after flowering (DAF). Weighted gene co-expression network analysis (WGCNA) revealed eight co-expression modules, among which the turquoise module was significantly correlated with the isoflavone content (P < 0.001). This module shared 1591 overlapping genes, including 98 genes encoding transcription factors (TFs), with the DEGs at 60 DAF, and it was significantly enriched in the isoflavone biosynthetic pathway according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (P < 0.0001). On the basis of high eigengene connectivity and strong co-expression relationships with isoflavone synthase genes (IFS1/IFS2), seven high-confidence candidate TFs were identified for further functional validation. Transcriptional regulation analysis revealed that GmWRKY44-2 functioned as a repressor by binding and suppressing both the IFS1 and IFS2 promoters and that GmMYB183 also served as a repressor by binding and suppressing the IFS2 promoter. Consistent with these findings, the results of soybean hairy root transformation confirmed that GmWRKY44-2 and GmMYB183 serve as negative regulators of isoflavone biosynthesis. These findings establish a foundation for understanding the molecular mechanisms underlying TF-mediated isoflavone regulation, with implications for engineering soybean varieties with tailored isoflavone profiles.