The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very‐long‐chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn‐2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.     

Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil.

Lysophosphatidic acid acyltransferase acylates the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid, a precursor to triacylglycerol. A cDNA encoding lysophosphatidic acid acyltransferase was isolated from developing seeds of meadowfoam (Limnanthes alba alba). The cDNA encodes a 281-amino acid protein with a molecular mass of 32 kD. The cDNA was expressed in developing seeds of transgenic high-erucic-acid rapeseed (Brassica napus) using a napin expression cassette. Erucic acid was present at the sn-2 position of triacylglycerols from transgenic plants but was absent from that position of seed oil extracted from control plants. Trierucin was present in the transgenic oil. Alteration of the sn-2 erucic acid composition did not affect the total erucic acid content. These experiments demonstrate the feasibility of using acyltransferases to alter the stereochemical composition of transgenic seed oils and also represent a necessary step toward increasing the erucic acid content of rapeseed oil.     

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F₁ plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.     

Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica

Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.     

Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds

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. Database: The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank under accession number DQ250814.     

Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds

The fatty acid composition in the seed oil was significantly modified following the introduction of transgenes. To further enhance the desirable characteristics of rapeseed oil, it would be beneficial to develop a new approach for the simultaneous silencing of two or more target genes. Our goals in the current study were to (1) increase oleic acid to more than 75%, (2) reduce polyunsaturated fatty acids (PUFA) to about 10% and erucic acid to zero, and (3) accomplish these changes in a single-transformation event. In a single transformation, two fragments amplified from the fatty acid ^Δ12-desaturase 2 (BnaFAD2) and fatty acid elongase 1 (BnaFAE1) genes of Brassica napus were linked together to form a fusion fragment. The fusion fragment was then used to assemble unique intron-spliced hairpin interfering constructs. In the transgenic plant FFRP4-4, the expression of BnaFAD2 and BnaFAE1 genes was completely inhibited. The composition of oleic acid in FFRP4-4 rose to 85%, PUFA dropped to 10% and erucic acid was undetectable. All hybrid F₁ seeds obtained from the reciprocal crossing of FFRP4-4 and GX-parents (with different genetic backgrounds) contained more than 80% oleic acid, about 10% PUFA and very low, or undetectable, erucic acid. The results confirmed that the fusion fragment silencing construct can simultaneously and effectively silence the target genes on a consistent basis. The strategy provides a useful tool for detecting gene function and advancing genetic engineering techniques for the improvement of agricultural crops.     

Assessment of FAE1 polymorphisms in three Brassica species using EcoTILLING and their association with differences in seed erucic acid contents

Background  

FAE1 (fatty acid elongase1) is the key gene in the control of erucic acid synthesis in seeds of Brassica species. Due to oil with low erucic acid (LEA) content is essential for human health and not enough LEA resource could be available, thus new LEA genetic resources are being sought for Brassica breeding. EcoTILLING, a powerful genotyping method, can readily be used to identify polymorphisms in Brassica.     

Towards the genetic engineering of triacylglycerols of defined fatty acid composition: major changes in erucic acid content at the sn-2 position affected by the introduction of a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii into oil seed rape

A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.     

Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.     

Mapping the genome of rapeseed (Brassica napus L.). II. Localization of genes controlling erucic acid synthesis and seed oil content

A F₁ microspore-derived DH population, previously used for the development of a rapeseed RFLP map, was analysed for the distribution of erucic acid and seed oil content. A clear three-class segregation for erucic acid content could be observed and the two erucic acid genes of rapeseed were mapped to two different linkage groups on the RFLP map. Although the parents of the segregating DH population showed no significant difference in seed oil content, in the DH population a transgressive segregation in oil content was observed. The segregation closely followed a normal distribution, characteristic of a quantitative trait. Using the program MAPMAKER/QTL, three QTLs for seed oil content could be mapped on three different linkage groups. The additive effects of these QTLs explain about 51% of the phenotypic variation observed for this trait in the DH population. Two of the QTLs for oil content showed a close association in location to the two erucic acid genes, indicating a direct effect of the erucic acid genes on oil content.     

Dissection of genetic architecture for glucosinolate accumulations in leaves and seeds of Brassica napus by genome‐wide association study

Glucosinolates (GSLs), whose degradation products have been shown to be increasingly important for human health and plant defence, compose important secondary metabolites found in the order Brassicales. It is highly desired to enhance pest and disease resistance by increasing the leaf GSL content while keeping the content low in seeds of Brassica napus, one of the most important oil crops worldwide. Little is known about the regulation of GSL accumulation in the leaves. We quantified the levels of 9 different GSLs and 15 related traits in the leaves of 366 accessions and found that the seed and leaf GSL content were highly correlated (r = 0.79). A total of 78 loci were associated with GSL traits, and five common and eleven tissue‐specific associated loci were related to total leaf and seed GSL content. Thirty‐six candidate genes were inferred to be involved in GSL biosynthesis. The candidate gene BnaA03g40190D (BnaA3.MYB28) was validated by DNA polymorphisms and gene expression analysis. This gene was responsible for high leaf/low seed GSL content and could explain 30.62% of the total leaf GSL variation in the low seed GSL panel and was not fixed during double‐low rapeseed breeding. Our results provide new insights into the genetic basis of GSL variation in leaves and seeds and may facilitate the metabolic engineering of GSLs and the breeding of high leaf/low seed GSL content in B. napus.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Down‐regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil

High oleic oil is an important industrial feedstock that has been one of the main targets for oil improvement in a number of oil crops. Crambe (Crambe abyssinica) is a dedicated oilseed crop, suitable for industrial oil production. In this study, we down‐regulated the crambe fatty acid desaturase (FAD) and fatty acid elongase (FAE) genes for creating high oleic seed oil. We first cloned the crambe CaFAD2, CaFAD3 and CaFAE1 genes. Multiple copies of each of these genes were isolated, and the highly homologous sequences were used to make RNAi constructs. These constructs were first tested in Arabidopsis, which led to the elevated oleic or linoleic levels depending on the genes targeted, indicating that the RNAi constructs were effective in regulating the expression of the target genes in nonidentical but closely related species. Furthermore, down‐regulation of CaFAD2 and CaFAE1 in crambe with the FAD2–FAE1 RNAi vector resulted in even more significant increase in oleic acid level in the seed oil with up to 80% compared to 13% for wild type. The high oleic trait has been stable in subsequent five generations and the GM line grew normally in greenhouse. This work has demonstrated the great potential of producing high oleic oil in crambe, thus contributing to its development into an oil crop platform for industrial oil production.     

QTL for phytosterol and sinapate ester content in <Emphasis Type="Italic">Brassica napus</Emphasis> L. collocate with the two erucic acid genes

Improving oil and protein quality for food and feed purposes is an important goal in rapeseed (Brassica napus L.) breeding programs. Rapeseed contains phytosterols, used to enrich food products, and sinapate esters, which are limiting the utilization of rapeseed proteins in the feed industry. Increasing the phytosterol content of oil and lowering sinapate ester content of meal could increase the value of the oilseed rape crop. The objective of the present study was to identify quantitative trait loci (QTL) for phytosterol and sinapate ester content in a winter rapeseed population of 148 doubled haploid lines, previously found to have a large variation for these two traits. This population also segregated for the two erucic acid genes. A close negative correlation was found between erucic acid and phytosterol content (Spearman’s rank correlation, r _s = −0.80^**). For total phytosterol content, three QTL were detected, explaining 60% of the genetic variance. The two QTL with the strongest additive effects were mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. For sinapate ester content four QTL were detected, explaining 53% of the genetic variance. Again, a close negative correlation was found between erucic acid and sinapate ester content (r _s = −0.66^**) and the QTL with the strongest additive effects mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. The results suggests, that there is a pleiotropic effect of the two erucic acid genes on phytosterol and sinapate ester content; the effect of the alleles for low erucic acid content is to increase phytosterol and sinapate ester content. Possible reasons for this are discussed based on known biosynthetic pathways. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Zero erucic acid trait of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) results from a deletion of four base pairs in the <Emphasis Type="Italic">fatty acid elongase 1</Emphasis> gene

The fatty acid elongase 1 (FAE1) gene is a key gene in the erucic acid biosynthesis in rapeseed. The complete coding sequences of the FAE1 gene were isolated separately from eight high and zero erucic acid rapeseed cultivars (Brassica napus L.). A four base pair deletion between T1366 and G1369 in the FAE1 gene was found in a number of the cultivars, which leads to a frameshift mutation and a premature stop of the translation after the 466th amino acid residue. This deletion was predominantly found in the C-genome and rarely in the A-genome of B. napus. Expression of the gene isoforms with the four base pair deletion in a yeast system generated truncated proteins with no enzymatic activity and could not produce very long chain fatty acids as the control with an intact FAE1 gene did in yeast cells. In the developing rape seeds the FAE1 gene isoforms with the four base pair deletion were transcribed normally but failed to translate proteins to form a functional complex. The four base pair deletion proved to be a mutation responsible for the low erucic acid trait in rapeseed and independent from the point mutation reported by Han et al. (Plant Mol Biol 46:229–239, 2001). Gang Wu, Yuhua Wu contribute equally to this article.