Functional Characterization of Soybean Diacylglycerol Acyltransferase 3 in Yeast and Soybean

Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to -3 of diacylglycerol (DAG) to form TAG. Here, two members of the gene family, and , were identified from the soybean ( ) genome. Both of them were predicted to encode soluble cytosolic proteins containing the typical thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed that expression was much higher than in various soybean tissues such as leaves, flowers, and seeds. Functional complementation assay using TAG-deficient yeast ( ) mutant H1246 demonstrated that GmDGAT3-2 fully restored TAG biosynthesis in the yeast and preferentially incorporated monounsaturated fatty acids (MUFAs), especially oleic acid (C18:1) into TAGs. This substrate specificity was further verified by fatty-acid feeding assays and enzyme activity characterization. Notably, transgenic tobacco ( ) data showed that heterogeneous expression of resulted in a significant increase in seed oil and C18:1 levels but little change in contents of protein and starch compared to the EV-transformed tobacco plants. Taken together, GmDGAT3-2 displayed a strong enzymatic activity to catalyze TAG assembly with high substrate specificity for MUFAs, particularly C18:1, playing an important role in the cytosolic pathway of TAG synthesis in soybean. The present findings provide a scientific reference for improving oil yield and FA composition in soybean through gene modification, further expanding our knowledge of TAG biosynthesis and its regulatory mechanism in oilseeds.