Integrated GWAS and Functional Analyses Reveal a Multi-Layer Regulatory Network Orchestrating Spermidine Homeostasis and Stress Tolerance in Soybean ( Glycine max (L.) Merr.)

Abstract Spermidine (Spd) is a pivotal polyamine involved in plant growth and stress tolerance, but its genetic regulation in soybeans (Glycine max) remains unclear. In this study, we quantified seed Spd content in 208 soybean accessions across two years by RP-HPLC and performed GWAS. Spd showed continuous variation and a high broad-sense heritability (H2 = 0.99). GWAS identified a major repeated locus on chromosome 18, and integrated haplotype and functional analyses prioritized GmSHMT, GmMYB91, and GmCAT9. Favorable haplotypes of these genes were associated with higher Spd content. Promoter analysis revealed common stress-responsive cis elements, and Y1H and dual-LUC assays showed that the MYB transcription factor Glyma.20G223300 directly binds and activates the GmSHMT promoter. Coexpression and RT-qPCR analyses indicated coordinated regulation of Spd biosynthetic genes. Y2H assays further revealed that GmSHMT physically interacts with GmMAT3 and GmSPDS1. Under drought and salt stress, candidate gene expression correlated with Spd accumulation, and favorable haplotypes conferred higher survival. Stable transgenic soybean lines overexpressing GmSHMT showed increased Spd content and enhanced drought and salt tolerance. These findings define a multilayer regulatory network controlling Spd homeostasis and provide genetic targets for stress-resilient soybean breeding.