Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 µm Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wild-type DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans.

ABSTRACT Soybean [ Glycine max (L.) Merr.] oil is one of the most consumed and highest produced vegetable oils. However, the high percentage of polyunsaturated fatty acids in soybean oil limits its stability and shelf life. Here we report the generation and characterization of a high oleic acid soybean event (305423 soybean) that has an elevated content of monounsaturated oleic acid and reduced presence of polyunsaturated linoleic acid and linolenic acid. This transgenic event was generated by the insertion of a soybean ω‐6 desaturase ( FAD2 ) gene fragment ( gm‐fad2‐1 ), resulting in the suppression of endogenous FAD2‐1 expression, and a modified version of the soybean acetolactate synthase ( ALS ) gene ( gm‐hra ), used as a selectable marker. Molecular characterization revealed that 305423 soybean harbors four DNA inserts containing multiple copies of complete and partial gm‐fad2‐1 gene cassettes and a single copy of the intact gm‐hra gene cassette. However, these DNA inserts seemed to be inherited together without segregation. The suppression of endogenous FAD2‐1 led to an increase of oleic acid (18:1) from 211 to 765 g kg –1 along with concurrent reduction of both linoleic acid (18:2) from 525 to 36.2 g kg –1 and linolenic acid (18:3) from 93.5 to 53.9 g kg –1 out of total fatty acyl groups. The 305423 soybean was also subjected to nutrient composition analyses and phenotypic and agronomic performance evaluations and found to be comparable to the control soybean except for the expected traits.

This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that genes encoding cytosolic pyrophosphatase enzymes provide novel targets to improve seed composition for plant biotechnology applications.

This chapter will take a broad look at non-acyl and acyl lipids found in seed. Initially it will focus on certain non-acyl lipids, such as sterols, carotenoids and tocochromanols. It will provide a brief overview of the function of these molecules in plants with a focus on seeds, review information on biosynthetic pathways and attempts at altering quality and quantity of these molecules through metabolic engineering. Our subsequent review of acyl lipid biosynthesis will mainly focus on recent findings related to modification of the pathways providing energy, reductant and carbon for fatty acid biosynthesis that allows for high levels of acyl lipid accumulation during seed filling. This section will have a specific focus on the interaction of primary metabolism and fatty acid biosynthesis. We will then briefly review fatty acid biosynthesis in seed plastids and cytosolic assembly of seed storage and membrane lipids in seed. Finally developmental regulation of seed storage lipid accumulation and transgenic approaches to increase the total oil content of seeds will be summarized.