Harnessing biotechnology and genetics to improve oilseed biochemistry

Abstract

With the push for alternative feedstock sources to replace fossil fuels in the near future due to rising concerns of climate change and environment, oilseed crops have emerged as an alternative because their vegetable oils are carbon neutral and biodegradable plus they can be implemented into multiple applications such as human food, animal feed, and different industrial products such as biodiesel and biolubricants. To achieve this goal, vegetable oils from oilseeds will need to have the optimal fatty acid profiles for the desired application and be able to have higher yields to keep up with global demand. First, a forward genetics approach was utilized to screen 1000 mutant Camelina sativa lines for fatty acid and or content changes. From this population of mutants, fatty acid knowledge in Arabidopsis thaliana was used to select potential gene candidates for mutations. Homeologs of FATTY ACID ELONGASE1 (FAE1), FATTY ACID DESATURASE2 (FAD2), FATTY ACID DESATURASE3 (FAD3), and [beta]-KETO-ACYL-ACP SYNTHASE II (KASII; FAB1) contained mutations that altered conserved amino acid residues in encoded proteins leading to lines with changes in fatty acid composition. Mutations of fae1c, fad2a, fae1a, and fad3a were crossed together to create a mid-oleic acid camelina seed oil. Seeds of this quadruple mutant had an approximate 40% oleic acid and reduced very long chain fatty acid (VLCFA) content in its oil. In addition, these fatty acid changes, led to increased oxidative stability. Next, a physical properties study on acetyl-triacylglycerols (acetyl-TAG) was conducted by blending purified acetyl-TAGs from transgenic expressing camelina seeds with Euonymus alatus DIACYLGLYCEROL ACETYLTRANSFERASE (EaDAcT) and with regular camelina triacylglycerols (TAG). Both TAGs were blended in different proportions and the resulting kinematic viscosity and thermal properties investigated. When 5 mol% increments of regular TAGs were added to the acetyl-TAG sample, the kinematic viscosity rose significantly. Interestingly, some of these blends were able to be within the kinematic viscosity limit for Diesel #4, thus allowing their use in low to medium speed engines. To study the effects of wounding stress on leaf lipids, a mass spectrometry-based screening approach of lipid extracts from wounded and unwounded leaves produced altered leaf phenotypes. A novel mutation in Arabidopsis thaliana FATTY ACID DESATURASE6 (FAD6) led to a decreased ratio of two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG (18:3/16:3) and MGDG (18:3/18:3) in leaf lipids. This mutation, fad6-3, had 13 amino acid deletion in its C-terminus and it was shown to lack enzymatic activity in yeast cells. In Arabidopsis, AT2G19450 (DIACYLGLYCEROL ACYLTRANSFERASE1, DGAT1) is the major enzyme that synthesizes triacylglycerols (TAGs) during seed development. The dgat1 mutant seeds possess low oil content in addition to a high polyunsaturated fatty acid (PUFA) composition. In addition, two desaturases AT3G12120 (FATTY ACID DESATURASE2, FAD2) and AT2G29980 (FATTY ACID DESATURASE3, FAD3) localized to the endoplasmic reticulum, were found to be highly upregulated in dgat1 developing seeds. FAD2 and FAD3 desaturate oleic acid (18:1) into linoleic acid (18:2) and 18:2 into alpha linolenic acid (18:3), respectively, at the sn-2 position on the glycerol backbone of PC molecules to form PUFA-PC. To investigate the flux of PUFA to DAG and PC, Arabidopsis lines lacking genes known to be involved in PUFA metabolism were generated. No double mutants of dgat1 fad2-1 were detected among either the F2 or F3 progeny. Reciprocal backcrosses with wild-type demonstrated that both the male and female dgat1 fad2-1 gametophytes are viable. Siliques from DGAT1/dgat1-1 fad2-1/fad2-1 and dgat1-1/dgat1-1 FAD2/fad2-1 have a 3:1 ratio of normal to aborted developing seeds indicating that the double mutant seeds are produced, but fail to fully mature. In contrast, mutant crosses of dgat1 fad3-2 produced viable double mutants. These homozygous double mutant seeds contained higher levels of 18:2, lower levels of VLCFAs, and lower fatty acid content levels compared to wild-type seeds. One hypothesis to explain these results is that in the absence of DGAT1 activity, desaturation of fatty acids by FAD2 becomes essential to provide PUFA substrates for PDAT1 to function. In a dgat1 fad2-1 mutant, seed development is arrested because TAG is unable to be synthesized by either DGAT1 or PDAT1. This can be further tested by lipid analysis on the aborted embryos.