AUTHOR=Xue Jinai , Gao Huiling , Xue Yinghong , Shi Ruixiang , Liu Mengmeng , Han Lijun , Gao Yu , Zhou Yali , Zhang Fei , Zhang Haiping , Jia Xiaoyun , Li Runzhi 

TITLE=Functional Characterization of Soybean Diacylglycerol Acyltransferase 3 in Yeast and Soybean

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.854103

DOI=10.3389/fpls.2022.854103

ISSN=1664-462X

ABSTRACT=<p>Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in <italic>de novo</italic> biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to <italic>sn</italic>-3 of diacylglycerol (DAG) to form TAG. Here, two members of the <italic>type 3 DGAT</italic> gene family, <italic>GmDGAT3-1</italic> and <italic>GmDGAT3-2</italic>, were identified from the soybean (<italic>Glycine max</italic>) genome. Both of them were predicted to encode soluble cytosolic proteins containing the typical thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed that <italic>GmDGAT3-2</italic> expression was much higher than <italic>GmDGAT3-1's</italic> in various soybean tissues such as leaves, flowers, and seeds. Functional complementation assay using TAG-deficient yeast (<italic>Saccharomyces cerevisiae</italic>) 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 <italic>in vitro</italic> enzyme activity characterization. Notably, transgenic tobacco (<italic>Nicotiana benthamiana</italic>) data showed that heterogeneous expression of <italic>GmDGAT3-2</italic> 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.</p>