Soybean (Glycine max (L.) Merr) is the largest oil and protein crop in the world and it is grown for both oil and protein. To address the needs of both the edible oil market and industrial applications of soybean oil, fatty acid modification has been a focus of soybean breeding programs. Natural variation, mutagenesis and genetic engineering have been used to alter the fatty acid profile. Several genes, mostly desaturases, have been associated with altered fatty acid profiles but enzymes in the Kennedy Pathway have yet to be studied as another source of genetic variation for altering the fatty acid profiles. The Kennedy Pathway is also known as the oil producing pathway and consists of four enzymes: glycerol-3-phosphate acyltransferase (G3PAT); lysophosphatidic acid acyltransferase (LPAAT); phosphatidic acid phosphatase (PAP); and diacylglycerol acyltransferase 1 (DGAT1). The starting material for this pathway is glycerol-3-phosphate, which is produced from glycerol by glycerol kinase (GK), and the product of this pathway is triacylglycerol (TAG). The overall objective of this study was to elucidate the role that the Kennedy Pathway plays in determining the fatty acid profile in two ways: (1) sequencing the transcribed region of the genomic genes encoding the enzymes of GK, G3PAT, LPAAT, and DGAT1 in soybean genotypes with altered fatty acid profiles; and (2) studying their expression over seed development, across three growing temperatures. The genetic material for the study consisted of four soybean genotypes with altered fatty acid profile: RG2, RG7, RG10, and SV64-53. Results from sequencing showed that the mutations identified in G3PAT, LPAAT, and DGAT1 in the four soybean genotypes did not explain the differences in the fatty acid profiles. The expression of G3PAT, LPAAT, and DGAT1 over seed development showed that G3PAT had the lowest levels, followed by LPAAT, then DGAT1, across the growing temperatures. The differences in expression among genotypes corresponded to differences in fatty acid accumulation, suggesting that expression rather than genetic mutations in the transcribed region of the genes influenced the fatty acid profile of the genotypes in this study. In conclusion, the enzymes of the Kennedy Pathway appear to contribute to the altered fatty acid profiles observed in the soybean mutant genotypes. / Ontario Ministry of Economic Development and Innovation (formerly Ontario Ministry of Research and Innovation), BioCar Initiative, Grain Farmers of Ontario, SeCan

The biofuel market is dominated by ethanol and biodiesel derived from cellulosic and lipid-based biomass crops. This is largely due to the relatively low costs and reliability of production. At present, production of non-food plant-derived oils for biofuel production in the U.S. is minimal. A research team from the Commonwealth Scientific and Industrial Research Organization (CSIRO), an independent Australian federal government research institution, has developed an efficient transgenic system to engineer oil production in tobacco leaves. This novel system is comprised of multiple transgenes that direct the endogenous metabolic flux of oil precursors towards triacylglycerol (TAG) production. Additional genes were incorporated to store and protect the accumulated oil in vegetative tissues. Preliminary greenhouse tests by the CSIRO research group indicated an oil content of > 30% by dry weight (DW) in tobacco leaf lamina. Here we evaluated two transgenic lines against a non-transgenic control in 2017 and 2018 in greenhouse and field production systems. The 2017 pilot study showed that the high leaf-oil tobacco line was viable and will grow in the field in Kentucky. Chemical analyses revealed significantly higher oil content compared to the non-transgenic control despite several logistical setbacks. These promising discoveries prompted the deployment of additional transgenic line assessments and further data validation in 2018. Line evaluations in 2018 revealed that the LEC2:WRI1:DGAT:OLE transgenic line had the highest leaf oil content (≥ 19.3% DW-1) compared to both the WRI1:DGAT:OLE transgenic line (≤ 5.6% DW-1) and non-transgenic control (≤ 2.1% DW-1). The results of this research will contribute to the successful development of transgenic tobacco lines engineered to accumulate high concentrations of TAG in the leaves.