Background The Arabidopsis thaliana dgat1 mutant, AS11 , has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1 , PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis. Methods Biochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity. Results Through microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1 / lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11 . Conclusions Collectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn -2 moieties to the sn -3 position of the enlarged AS11 DAG pool.

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 Δ13), docosadienoic acid (22:2 Δ5, Δ13) and nervonic acid (24:1 Δ15); 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. Published in 2010 by John Wiley & Sons, Ltd.

Lesquerella fendleri (commonly known as “Fendler’s bladderpod” or “yellowtop”) is a member of the Brassicaceae and is an important seed oil-producing plant. The lipid profile of L. fendleri seed indicates potential for producing a high quality replacement for castor oil. In this work, characterization of the lipid content of a suspension cell culture, derived from seedlings of L.fendleri, is provided. Under the described suspension cell culture conditions, 16:0, 18:1Δ9, 18:2 Δ9, Δ12 and 18:3 Δ9, Δ12, Δ15 fatty acids were found to accumulate in the cells, while 16:0, 26:0 and 28:0 fatty acids were predominant in the culture medium. Subsequently, the effect of application of abscisic acid (ABA), which modulates lipid accumulation, was assessed. Exogenously applied ABA was taken up by the cells and metabolized via the conjugation pathway, resulting in the accumulation of ABA-glucose ester. Preliminary tests demonstrate the cell line is responsive to exogenous ABA, resulting in increased cellular lipid content and increased accumulation of lipids in the culture medium. This novel L. fendleri suspension culture presents a valuable model system for efficient characterization of mechanisms associated with ABA-induced accumulation of lipids.

Nervonic acid 24:1 Delta15 (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 T(3) 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.

The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabidopsis thaliana (L.) Heynh ecotype Columbia and Brassica napus ‘Jet Neuf’ and over-expressed them in canola under the control of the seed-specific promoter, napin. DGAT1 from A. thaliana was inserted into B. napus ‘Quantum,’ whereas DGAT1 from B. napus was introduced into the B. napus double haploid breeding line DH12075. Both sets of transgenic plants exhibited increased seed oil content in both greenhouse and in field trial settings, ranging from 2.5% to 7% of dried mass on an absolute basis. The ‘Quantum’ transgenic lines were field-tested in plots at Watrous, Saskatchewan, in 2006 and 2007. Larger scale field trials of the DH12075 transgenics were carried out in 2007 at Ellerslie and Vegreville, Alberta. This is the first study wholly dedicated to DGAT1 over-expression and the resultant oil-content increases in transgenic canola under field conditions. Collectively, the field trial results strongly support the hypothesis that the level of DGAT1 activity during seed development in an oilseed crop can have a substantial effect on the flow of carbon into seed oil. Therefore, the over-expression of DGAT1 is a positive strategy for increasing oil content and cultivar performance in canola.

Nervonic acid is a Very Long-Chain Monounsaturated Fatty Acid (VLCMFA), 24:1 Δ15 (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 “money plant” (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 7–10 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 20–30-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.

In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be essential for growth and metabolism of yeast cells, they may play an important role in cell survival by metabolizing excess intracellular lysophospholipids. To test our hypothesis, we overexpressed a plant phospholipase A2 (PLA2) in both WT and plb123 cells, producing a pool of lysophosphatidylcholine (lysoPtdCho) in both transformants. In ¹⁴C acetate labeling experiments, WT cells were able to catabolize the resultant labeled lysoPtdCho, preventing accumulation, and the cells grew normally. In contrast, in the triple plb123 mutant PLA2 transformant, lysoPtDCho accumulated more than 4-fold to a toxic level, inhibiting cell growth. However, this growth inhibition was complemented by co-expression of either PLB1, PLB2 or PLB3 in the plb123 triple mutant already expressing the plant PLA2. Furthermore, in labeling experiments, the rescued cells exhibited a 60-75% reduction in 14C-lysoPtdCho build-up compared to plb123PLA2 cells. Our data provides conclusive evidence that yeast PLBs can metabolize intracellular lysoPtdCho produced by plant PLA2 overexpression in yeast. Our experiments indicate the utility of ectopic plant phospholipase A gene expression to characterize poorly-understood phospholipid metabolism mutants in yeast or other organisms.

The 3′-UTR of the FAD2 gene from Brassica carinata was cloned by PCR and used to prepare an intron-spliced hairpin RNA (ihpRNA) construct. Compared to that of the wild type (WT) background, this construct, when expressed in B. carinata, resulted in a high degree of FAD2 gene silencing accompanied by strong increases of up to 16 and 10% in oleic acid and erucic acid proportions, respectively. The increase in 18:1 was accompanied by a concomitant proportional reduction in 18:2. A second construct containing ihpRNA targeted to the endogenous FAD2 gene in addition to the heterologous Crambe abyssinica FAE gene under the control of seed specific napin promoter, was used to transform B. carinata. This approach resulted in an even greater increase in erucic acid proportions, by up to 16% in T1 segregating seeds as compared to that of the WT control. To our knowledge, this is currently the highest accumulation of erucic acid achieved in B. carinata seeds using transgenic approaches, making it an increasingly-attractive alternative to high erucic B. napus cultivars as an industrial oil crop.

SUMMARY A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Deterioration of conifer seeds during prolonged storage has a negative impact on reforestation and gene conservation efforts. Western redcedar (Thuja plicata Donn ex D. Don) is a species of tremendous value to the forest industry. The seeds of this species are particularly prone to viability losses during long-term storage. Reliable tools to assess losses in seed viability during storage and their underlying causes, as well as the development of methods to prevent storage-related deterioration of seeds are needed by the forest industry. In this work, various imaging methods and biochemical analyses were applied to study deterioration of western redcedar seeds. Seedlots that exhibited poor germination performance, i.e. those that had experienced the greatest losses of viability during prolonged storage, exhibited greater abundance of oxidized proteins, detected by protein oxidation assays, and more pronounced changes in their in vivo (13)C NMR spectra, most likely due to storage oil oxidation. The proportion of oxidized proteins also increased when seeds were subjected to accelerated ageing treatments. Detection of oxidized oils and proteins may constitute a reliable and useful tool for the forest industry.

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Background: Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed.Results: Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.Conclusion: Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

A genomic fatty acid elongation 1 (FAE1) clone was isolated from Crambe abyssinica. The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20:1(delta11)) and erucic (22:1(delta13)) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content.

Using a PCR-based approach, a genomic clone of a putative fatty acid elongase (FAE) showing high similarity to plant microsomal fatty acid elongases, was isolated from Teesdalia nudicaulis. The genomic clone contains an open reading frame of 1521bp encoding a protein of 507 amino acids. Expression of the Teesdalia nudicaulis FAE homolog in yeast cells resulted in accumulation of eicosenoic (20:1Δ11) and 20:1Δ13, fatty acids not normally present in wild-type yeast. Northern blot analyses indicated that the Teesdalia nudicaulis FAE is expressed in seeds but not in vegetative tissues. Transformation of a low eicosenoic acid Arabidopsis thaliana mutant line (AC56) with the Teesdalia nudicaulis FAE restored elongase activity and the high proportion of eicosenoic acid typically found in wild-type Arabidopsis seed oils. The recombinant FAE expressed in a yeast transformant showed a strong preference for elongating an oleoyl to an eicosenoyl moiety in vitro.

Using a degenerate primers approach, a cDNA of a putative microsomal fatty acid desaturase showing high homology to other plant microsomal FAD2 (Δ12) desaturases was obtained from Tropaeolum majus. This cDNA contains an open reading frame of 1152bp encoding a protein of 383 amino acids. Expression of the T. majus FAD2 oleate-Δ12 desaturase homolog in yeast cells resulted in accumulation of an appreciable amount of cis-9,12-hexadecadienoic (16:2Δ9,12) and linoleic acid (18:2Δ9,12), fatty acids not normally present in wild type yeast, indicating that the gene encodes a functional FAD2 enzyme. Genetic analyses showed that the T. majus FAD2 is represented by at least one constitutively expressed gene. Transformation of a low linoleic (18:2Δ9,12) Arabidopsis mutant line (AL63) with the T. majus FAD2 cDNA restored oleate-Δ12 desaturase activity and wild type proportions of linoleate in the seed oils of the transgenic plants.

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1Δ5 and the diene 22:2Δ5,Δ13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2Δ5,Δ13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1Δ5 to approximately 10% of the total fatty acids of seeds. The content of C20 and C22 fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2Δ5,Δ11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C20 and C22 fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1Δ3, respectively.

Erucic acid and its derivatives represent important industrial feedstock compounds, and there is an increasing demand for the production of high erucate oils in this regard. Our goal therefore, is to develop high erucic acid (HEA) Brassicaceae lines with increased proportions of erucic acid and very long-chain fatty acids (VLCFAs). We proposed that oleate availability may be a rate-limiting factor in the biosynthesis of erucic acid. We have tried to address this question by manipulating the expression of the endogenous FAD2 gene in B. carinata using co-supression and antisense approaches. Both methods resulted in transgenic lines exhibiting decreased proportions of polyunsaturated C18 fatty acids (18:2+18:3) and concomitant and significantly increased proportions of 18:1, 22:1 and total VLCFAs. Co-suppressed FAD2 B. carinata lines exhibited 3–18% decreases in 18:2, 22–49% decreases in 18:3 and significantly increased proportions of 18:1 (36–99%), 22:1 (12–27%) and VLCFAs (6–15%). Transgenic B. carinata lines developed using an antisense FAD2 approach exhibited decreased proportions of 18:2 and 18:3 (9–39% and 33–48%, respectively) and significantly increased proportions of 18:1 (54–130%), 22:1 (5–19%) and VLCFAs (6–21%). The possibility of using these approaches to produce prototype transgenic germplasm of the Brassicaceae accumulating seed oils with improved proportions of erucic and other VLCFAs is discussed.

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.

To 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.

We have isolated fatty acid elongation 1 (FAE1) genomic clones from high erucic acid (HEA) B. napus, B. rapa and B. oleracea, and low erucic acid (LEA) B. napus. Nucleotide sequences corresponding to open reading frames of 1523 bp were translated and proteins of 506 a.a. were deduced. Comparative study of FAE1 protein sequences from HEA and LEA Brassicas revealed the one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus. Using site directed mutagenesis the phenylalanine(282) residue was substituted with a serine residue in FAE1 polypeptide from LEA B. napus, the mutated gene was expressed in yeast, and GC analysis showed presence of very long chain mono-unsaturated fatty acids indicating that the elongase activity was restored in the LEA FAE1 enzyme. Thus, for the first time, the low erucic acid trait in B. napus has been attributed to a single amino acid substitution which prevents the biosynthesis of very long chain mono-unsaturated fatty acids.