Functional Characterization of Soybean Diacylglycerol Acyltransferase 3 in Yeast and Soybean.

ABSTRACT

Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in de novo biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to sn-3 of diacylglycerol (DAG) to form TAG. Here, two members of the type 3 DGAT gene family, GmDGAT3-1 and GmDGAT3-2, were identified from the soybean (Glycine max) genome. Both of them were predicted to encode soluble cytosolic proteins containing the typical thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed that GmDGAT3-2 expression was much higher than GmDGAT3-1's in various soybean tissues such as leaves, flowers, and seeds. Functional complementation assay using TAG-deficient yeast (Saccharomyces cerevisiae) mutant H1246 demonstrated that GmDGAT3-2 fully restored TAG biosynthesis in the yeast and preferentially incorporated monounsaturated fatty acids (MUFAs), especially oleic acid (C181) into TAGs. This substrate specificity was further verified by fatty-acid feeding assays and in vitro enzyme activity characterization. Notably, transgenic tobacco (Nicotiana benthamiana) data showed that heterogeneous expression of GmDGAT3-2 resulted in a significant increase in seed oil and C181 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 C181, 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.