Genome-wide identification and characterization of the galactinol synthase (GmGolS) gene family and its expression under drought, heat, and combined stress in soybean.

Galactinol synthase (GolS) catalyzes the first committed step in raffinose family oligosaccharide (RFO) biosynthesis and is implicated in plant adaptation to abiotic stress. Although GolS gene families have been characterized in several plant species, a comprehensive genome-wide analysis in soybean has been lacking. Here, we identified 25 GmGolS genes in the soybean genome, ranging from 328 to 644 amino acids in length, and characterized their genomic organization, evolutionary history, and stress-responsive expression. The 25 GmGolS genes were distributed unevenly across 11 chromosomes, with chromosomes 3 and 19 showing the highest gene density. Phylogenetic analysis with orthologs from Arabidopsis thaliana and Oryza sativa classified all GolS proteins into six groups, and structural conservation within groups suggests shared functional ancestry. Synteny analysis indicated that segmental duplication, supported by tandem clusters on chromosomes 4 and 5, appears to be the predominant mechanism underlying family expansion, with duplication events estimated to span from approximately 0.20 to 112.87 million years ago. Ka/Ks analysis of duplicate gene pairs indicated values predominantly below 1, consistent with purifying selection acting on this family since duplication. Subcellular localization predictions suggested diverse targeting to chloroplasts, the cytoplasm, and the plasma membrane, pointing to potential multi-compartment roles, while protein-protein interaction network analysis linked GmGolS proteins to carbohydrate-metabolism and stress-responsive partners, and homology modeling indicated a conserved GT8 fold consistent with the catalytic requirements for galactinol synthesis. To examine stress-responsive expression, RT-qPCR was performed on three soybean cultivars subjected to drought, heat, and combined stress at the seedling stage. GmGolS genes displayed variable and stress-specific expression patterns, with combined stress eliciting the strongest transcriptional response overall. GmGolS-3, GmGolS-9, GmGolS-10, GmGolS-13, and GmGolS-17 showed comparatively higher expression across treatments, with GmGolS-3 and GmGolS-9 most strongly induced under combined stress; by contrast, GmGolS-11 responded primarily to heat stress alone, and GmGolS-13 was consistently downregulated across all treatments, suggesting functional divergence within the family. Taken together, these findings provide a foundation for understanding the evolution and stress-responsive functions of the GmGolS gene family in soybean and identify GmGolS-3 and GmGolS-9 as candidates warranting further investigation, though functional validation is required to confirm the precise biological roles of individual members.