There is growing interest in expanding the production of soybean oils (mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastid‐localized glycerol‐3‐phosphate dehydrogenase (GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol‐3‐phosphate (G3P), an obligate substrate required for TAG biosynthesis. Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids (FAs), especially oleic acid (C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3P content led to an elevated diacylglycerol (DAG) pool, in which the Kennedy pathway‐derived DAG was mostly increased, followed by PC‐derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in‐line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1‐mediated G3P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.

Soybean is a major crop that provides oil and protein worldwide. Soybean products have been consumed for centuries in many different kinds of food. The regulatory network that controls the accumulation of fatty acids (FAs) and storage proteins (SSPs) in seeds is poorly understood. To gain new insights into the molecular mechanisms that contribute to high‐quality seeds, seed FA and SSP contents were analyzed for the effects of chromosome segment substitution in a wild soybean line (CSSL). High‐throughput transcriptomics and tandem mass tag (TMT)‐based quantitative proteomics were performed on the dry seeds of the CSSL and parent lines. In total, 665 differentially expressed genes (DEGs) and 83 differentially accumulated proteins (DAPs) were identified. Of these 27 DEGs and 23 DAPs were found to regulate the seed storage profile. These genes encode proteins involved in photosynthesis, protein processing, protein sorting, and storage protein accumulation. Data are presented showing that FA synthesis was decreased by the regulation of SSP‐accumulation‐related genes and proteins. Taken together, the results provide new insights into the regulation network involved in the accumulation of seed storage compounds involving both source and sink processes that may be used as markers in future soybean breeding programs.