Characterization of the Soybean GPAT Gene Family Identifies GmGPAT1 as a Key Protein in Salt Stress Tolerance

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial and rate-limiting step of glycerolipid biosynthesis, yet their contribution to salt tolerance in the soybean (<i>Glycine max</i> (L.) Merr.) plants remains largely uncharacterized. In this study, a total of 27 <i>GmGPAT</i> genes were identified, and their evolutionary relationships, chromosomal distribution, conserved motifs, and cis-regulatory elements were comprehensively analyzed. Through transcriptomic and qPCR analyses, many <i>GmGPATs</i> were found to be predominantly expressed in roots, with <i>GmGPAT1</i>, a plastid-targeted isoform, displaying the most rapid and pronounced transcriptional activation under salt stress. GFP-fusion experiments in transient expression assays confirmed plastid localization of <i>GmGPAT1</i>. Heterologous expression in <i>Escherichia coli</i> together with enzyme kinetics analyses validated its enzymatic function as a GPAT family member. The soybean hairy-root lines overexpressing <i>GmGPAT1</i> exhibited enhanced root elongation, increased biomass, and improved photosynthetic efficiency under 120 mM NaCl stress, while CRISPR/Cas9 knockout mutants showed pronounced growth inhibition. Physiological assays demonstrated that <i>GmGPAT1</i> overexpression mitigated oxidative damage by limiting reactive oxygen species (ROS) accumulation and lipid peroxidation, increasing antioxidant enzyme activities (CAT, SOD, POD), and elevating the ratios of AsA/DHA and GSH/GSSG. These changes contributed to redox homeostasis and improved Na⁺ extrusion capacity. A genome-wide association study (GWAS) involving 288 soybean accessions identified a single nucleotide polymorphism in the <i>GmGPAT1</i> promoter that was significantly correlated with salt tolerance, and the beneficial <i>Hap1</i> allele emerged as a promising molecular marker for breeding. Together, these analyses emphasize the status of <i>GmGPAT1</i> as a major regulator of salt stress adaptation through the coordinated modulation of lipid metabolism and redox balance, extend the functional annotation of the soybean GPAT family, and highlight new genetic resources that can be leveraged to enhance tolerance to salt stress in soybean cultivars.