Oil bodies isolated from Brassica napus mature seed

Abstract only availablePlants store seed triacylglycerols in discrete lipid monolayer storage organelles called oil bodies. Only two proteins have been characterized from oil bodies, namely oleosin and caleosin, which are both integral membrane proteins. To better understand the protein composition this organelle, oil bodies were isolated from Brassica napus (cultivar westar) mature seed. Oil bodies were isolated using two published methods that utilize phase separation in aqueous media. Method 1 employed iterative washes in aqueous media containing sucrose, and 2M NaCl while method 2 made use of only one type of aqueous media (minus NaCl) put through multiple washes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed method 1 to yield isolated oil bodies with higher purity based on the absence of the storage protein napin that was present in the total protein of isolated oil bodies from method 2. The oil bodies isolated with method 1 were subjected to washes in 2M NaCl or 8M urea to determine the nature of protein association to oil bodies. The isolated oil bodies were fractionated through petroleum ether to extract neutral lipids (triacylglycerols) that are contained by the monolayer membrane. Polar lipids were then extracted with chloroform/methanol. The interfacial pad which contained the associated proteins was suspended in water, sonicated, and subjected to acetone precipitation. Analysis of salt and urea washed oil body proteins by SDS-PAGE revealed abundant bands of the proper molecular weight for oleosins as well as at least 10 other additional proteins. Identification of these proteins by mass spectrometry will reveal novel proteins associated with oil bodies.Plant Genomics Internship @ M

Gaining insight into the role of serine 282 in B. napus FAE1 condensing enzyme

AbstractTo gain some insight whether there is an absolute requirement for the serine 282 to yield a functional fatty acid elongase 1 condensing enzyme we have introduced point mutations in the FAE1 coding sequence which led to the substitution of serine 282 with several aliphatic or aromatic amino acids. The mutated FAE1 polypeptides were expressed in yeast. Gas chromatography analyses of the fatty acid methyl esters from yeast lysates and fatty acid elongase activity assays demonstrated that there is not an absolute requirement for serine at position 282 to yield a functional FAE1 condensing enzyme

Increase of Nervonic Acid Content in Transformed Yeast and Transgenic Plants by Introduction of a Lunaria annua L. 3-Ketoacyl-CoA Synthase (KCS) Gene

Nervonic acid is a Very Long-Chain Monounsaturated Fatty Acid (VLCMFA), 24:1 \u39415 (cis-tetracos-15-enoic acid) found in the seed oils of Lunaria annua, borage, hemp, Acer (Purpleblow maple) and Tropaeolum speciosum (Flame flower). However, of these, only the \u201cmoney plant\u201d (Lunaria annua L.) has been studied and grown sparingly for future development as a niche crop and the outlook has been disappointing. Therefore, our goal was to isolate and characterize strategic new genes for high nervonic acid production in Brassica oilseed crops. To this end, we have isolated a VLCMFA-utilizing 3-Keto-Acyl-CoA Synthase (KCS; fatty acid elongase; EC 2.3.1.86) gene from Lunaria annua and functionally expressed it in yeast, with the recombinant KCS protein able to catalyze the synthesis of several VLCMFAs, including nervonic acid. Seed-specific expression of the Lunaria KCS in Arabidopsis resulted in a 30-fold increase in nervonic acid proportions in seed oils, compared to the very low quantities found in the wild-type. Similar transgenic experiments using B. carinata as the host resulted in a 7\u201310 fold increase in seed oil nervonic acid proportions. KCS enzyme activity assays indicated that upon using 14C-22:1-CoA as substrate, the KCS activity from developing seeds of transgenic B. carinata was 20\u201330-fold higher than the low erucoyl-elongation activity exhibited by wild type control plants. There was a very good correlation between the Lun KCS transcript intensity and the resultant 22:1-CoA KCS activity in developing seed. The highest nervonic acid level in transgenic B. carinata expressing the Lunaria KCS reached 30%, compared to 2.8% in wild type plant. In addition, the erucic acid proportions in these transgenic lines were considerably lower than that found in native Lunaria oil. These results show the functional utility of the Lunaria KCS in engineering new sources of high nervonate/reduced erucic oils in the Brassicaceae.Peer reviewed: YesNRC publication: Ye

Seed-Specific Heterologous Expression of a Nasturtium FAE Gene in Arabidopsis Results in a Dramatic Increase in the Proportion of Erucic Acid

The fatty acid elongase [often designated FAE or β-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium β-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed

Molecular Cloning and Characterization of a KCS gene from Cardamine graeca and its Heterologous Expression in Brassica Oilseeds to Engineer High Nervonic Acid Oils for Potential Medical and Industrial Use

Nervonic acid 24:1 \u39415 (cis-tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid. There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells. We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T3 generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic/highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical/nutraceutical applications to improve human and animal health.Peer reviewed: YesNRC publication: Ye

Improving euricic acid content in rapeseed through biotechnology: what can the Arabidopsis FAE1 and the Yeast SLC1-1 genes contribute

The main goal of our research is to produce, by genetic manipulation, Brassica napus L. cultivars with higher amounts of 22:1 in their seed oil than in present Canadian high erucic acid rapeseed (HEAR) cultivars developed through traditional breeding, ideally with proportions of 22:1 approaching 80 mol% (828 g kg\u207b\ub9). To probe some rate-limiting steps in the accumulation of triacylglycerols containing very long chain fatty acids (VLCFAs), particularly erucic acid (22:1), we have taken a transgenic approach, studying the effect of expressing two target genes in HEAR B. napus cv. Hero. To study the role of the elongase complex, involved in elongation of C18 fatty acid moieties to produce VLCFAs, we expressed the Arabidopsis thaliana L., fatty acid elongase 1 (FAE1) gene under the control of a seed-specific promoter (napin), in Hero. This resulted in increased proportions of 22:1 in the seed oil, rising from 430 g kg\u207b\ub9 in non-transformed controls to 480 to 530 g kg\u207b\ub9 22:1 in FAE1 transgenic Hero lines. The FAE1 lines exhibited higher elongase activity in vitro compared to control lines. These data suggest that the level of active condensing enzyme in the native elongase complex is somewhat rate limiting for synthesis of erucic acid and other VLCFAs in HEAR. In small scale field trials, the VLCFA and 22:1 content of FAE1 transgenic lines were superior to field-grown control lines. We report that in field plot trials, the progeny of our best T4 B. napus cv. Hero SLC1-1 transgenic lines clearly out-performed controls in terms of 22:1, oil content, and yield.Peer reviewed: YesNRC publication: Ye

Brassica carinata-A New Molecular Farming Platform for Delivering Bio-industrial Oil Feedstocks: Case studies of Genetic Modifications to Improve Seed Very Long-Chain Fatty Acid and Oil Content in Seeds

Crop development and species diversity are important aspects of the emerging global bioeconomy, as is maximizing crop value through total crop utilization. We advocate development of Brassica carinata as a biorefinery and bioindustrial oils platform using traditional and molecular breeding techniques and tools. We review genetic studies and breeding efforts to develop elite B. carinata germplasm, work involving development of transformation and regeneration protocols, target gene isolation, and transgene expression. Genetic modification strategies using a B. carinata breeding line as a delivery platform for very long-chain fatty acid-enhanced/modified oils are presented as case studies. The target oil products are erucic acid (22:1 \u39413), docosadienoic acid (22:2 \u3945, \u39413) and nervonic acid (24:1 \u39415); in addition transgenic efforts to enhance B. carinata seed oil content are discussed. The overall advantages and current limitations to utilizing this crop are delineated. Other anticipated biobased products from a B. carinata platform may include, but are not limited to, the production of biolubricants, biofuels and biopolymers from the oil, biopesticides, antioxidants, as well as plant gums, and vegetable protein-based bioplastics and novel food and feed products. In summation, this collaborative B. carinata breeding/germplasm development/value-added molecular modification effort will not only contribute to the development of renewable feedstocks for the emerging Canadian bioeconomy (biorefinery/bioproducts), but also promises to generate positive economic and environmental benefits.Peer reviewed: YesNRC publication: Ye