A Novel Soybean Diacylglycerol Acyltransferase 1b Variant with Three Amino Acid Substitutions Increases Seed Oil Content

Abstract

Improving soybean (Glycine max) seed composition by increasing the protein and oil components will add significant value to the crop and enhance environmental sustainability. Diacylglycerol acyltransferase (DGAT) catalyzes the final rate-limiting step in triacylglycerol biosynthesis and has a major impact on seed oil accumulation. We previously identified a soybean DGAT1b variant modified with 14 amino acid substitutions (GmDGAT1b-MOD) that increases total oil content by 3 percentage points when overexpressed in soybean seeds. In the present study, additional GmDGAT1b variants were generated to further increase oil with a reduced number of substitutions. Variants with one to four amino acid substitutions were screened in the model systems Saccharomyces cerevisiae and transient Nicotiana benthamiana leaf. Promising GmDGAT1b variants resulting in high oil accumulation in the model systems were selected for overexpression in soybeans. One GmDGAT1b variant with three novel amino acid substitutions (GmDGAT1b-3aa) increased total soybean oil to levels near the previously discovered GmDGAT1b-MOD variant. In a multiple location field trial, GmDGAT1b-3aa transgenic events had significantly increased oil and protein by up to 2.3 and 0.6 percentage points, respectively. The modeling of the GmDGAT1b-3aa protein structure provided insights into the potential function of the three substitutions. These findings will guide efforts to improve soybean oil content and overall seed composition by CRISPR editing.

Keywords: Glycine max; DGAT1; Lipid; Oil; Protein; Seed composition; Soybean.

Fig. 1

Evaluation of GmDGAT1b variants in model systems. (A) Nile red staining of GmDGAT1b variants expressed in the S. cerevisiae DGA1Δ/LRO1Δ double mutant strain. At least four biological replicates and six technical replicates were analyzed for each variant. Error bars denote standard deviation. (B) Total oil accumulation in N. benthamiana leaves expressing GmDGAT1b variants. Six biological replicates were analyzed for each variant. Individual data points are represented as dots, and error bars represent the standard error of the mean. One-way ANOVA revealed that statistically significant differences were present between at least two groups (F = 26.3, P < 0.0001). Tukey’s Honestly Significant Difference test was performed for pairwise comparisons between all gene variants, with non-overlapping letters signifying statistically significant differences (P < 0.05).

Fig. 2

T₃ seed analysis of GmDGAT variants expressed using the oleosin promoter. Seed was analyzed by FT-NIR spectroscopy for oil (A) and protein (B), with n ≥ 6 representing at least two events per variant. Oil and protein values are reported on a wt%, at 13% moisture, basis. Statistical comparisons of seeds expressing each GmDGAT1b variant were made with respect to sibling null segregants by Student’s t-test, with asterisks representing significant differences (P < 0.05).

Fig. 3

T₃ seed analysis of GmDGAT1b-3aa and GmDAT1b MOD (soil 111) expressed using the β-conglycinin promoter. Seed was analyzed by NIT spectroscopy for oil (A) and protein (B) (n ≥ 6). Oil and protein values are reported on a wt%, at 13% moisture, basis. Statistical comparisons for each event were made with respect to null segregants by Student’s t-test, with asterisks representing significant differences (P < 0.05).

Fig. 4

Structural analysis of the GmDGAT1b-3aa variant modeled by AlphaFold. (A) Overall predicted structure represented as a dimer. (B and C) (two views) position of the three amino acid substitutions (yellow) in the GmDGAT1b-3aa variant, putative active site (red), acyl-CoA binding sites (blue) and the conserved FYXDWWN motif (green).