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 type 3 DGAT gene family, GmDGAT3-1 and GmDGAT3-2, were identified from 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 (C18:1) into TAGs. This substrate specificity was further verified by feeding assays and in vitro enzyme activity characterization. Notably, transgenic tobacco (Nicotiana benthamiana) data showed that heterogeneous expression of GmDGAT3-2 resulted in significant increase of 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.

These two GmDGAT3 genes were identified from the soybean genome in Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html#!info? Alias =Org _Gmax). 

The accession number of GmDGAT3-1 is Glyma.13G118300.1 (https://phytozome-next.jgi.doe.gov/report/gene/Gmax_ Wm82_a2_v1/Glyma.13G118300).

The accession number of GmDGAT3-2 is Glyma.17G041600.1 (https://phytozome-next.jgi.doe.gov/report/gene/Gmax_Wm82_a2_v1/Glyma.17G041600 )

A phylogenetic tree of GmDGAT3 and other DGAT3 proteins was constructed using the neighbor-joining method in the MEGA 6.0 (see Table S1 for protein information derived from different species and the sequence downloaded from NCBI (https://www.ncbi.nlm.nih.gov) ).

All experiments were carried out at least in triplicate. The data was analyzed using IBS SPSS Statistics software Version 22. Duncan's test was employed to detect the statistical significance (P < 0.05) when comparing differences between two groups.