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.     

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.     

Detection of induced mutations in CaFAD2 genes by next‐generation sequencing leading to the production of improved oil composition in Crambe abyssinica

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down‐regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS‐mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one‐dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non‐sense mutations and mis‐sense mutations in CaFAD2‐C1, ‐C2 and ‐C3. The homozygous non‐sense mutants for CaFAD2‐C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis‐sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non‐sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.     

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%–50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%–60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild‐type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine‐diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.     

Fatty acid composition of the cold-water-inhabiting freshwater red alga Sirodotia Kylin

Four samples of freshwater alga Sirodotia (class Rhodophyceae) collected from two distinct streams in the Mahabaleshwar, Satara district (1,732 m a.s.l.) of the Western Ghats of Maharashtra (India) were analysed for their fatty acid content. The presence of 32 fatty acids was revealed, of which 13 were saturated (SFA), 8 were monounsaturated (MUFA) and 11 were polyunsaturated (PUFA) fatty acids. The major finding was the presence of three pharmaceutically and neutraceutically important PUFAs: arachidonic acid (AA), eicosapentanoeic acid (EPA), and docosahexanoiec acid (DHA). The major fatty acids identified were palmitic (16:0), cis-11,14 icodienoic (20:2), behenic (22:0), cis-8,11,14 eicosatrienoic(20:3n6), cis-4,7,10,13,16,19 docosahexanoeic (22:6n3), cis-13,16 docosadienoic (22:2), erucic (22:1n9), -5,8,11,14,17 eicosapentaenoic (20:5n3), trichosonoic (23:0), nervonic (24:0), arachidonic (20:4n6), cis-10 pentadecanoic (15:1), cis-11,14,17 eicosatrienoic (20:3n3), and myristic acid (14:0). The total PUFA contents ranged from 31.45 to 40.37%. The fatty acids were characterised by the relatively high abundance of PUFAs, while C20 unsaturated acids were appreciably more abundant than C18 unsaturated acids. This is the first report on fatty acid profiles of the genus Sirodotia.     

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.     

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F₂ plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F₂ plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F₂ plants were confirmed in the next generation by analysing F₄ seeds harvested from five F₃ plants per selected F₂ plant. F₃ lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.     

Eliminating expression of erucic acid-encoding loci allows the identification of “hidden” QTL contributing to oil quality fractions and oil content in <Emphasis Type="Italic">Brassica juncea</Emphasis> (Indian mustard)

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2), fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.     

Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri

 Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F₂). Furthermore, crosses between the F₂ plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F₃ seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F₆ generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F₆ plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F₆ generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F₆ plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F₂ generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis. Received: 12 December 1998 / Accepted: 4 January 1999     

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.     

Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea FAT TY ACID ELONGATION1 (BjFAE1) gene

In Brassicas, the Fatty Acid Elongation1 (FAE1) gene product, a 3-ketoacyl-CoA synthase, is the first in a 4-enzyme complex involved in the synthesis of erucic acid from oleic acid. The FAE1 homologue from Brassica juncea cv. Pusa Bold was cloned in a binary vector both in sense and antisense orientations under the control of the CaMV35S promoter. The recombinant binary vectors were used to transform B. juncea cv. RLM 198 via Agrobacterium tumefaciens. The presence of the transgene was confirmed by polymerase chain reaction and Southern hybridization. Northern and western analyses showed the expression of the gene and protein, respectively, in the transgenic plants. Analyses of the fatty acid profile of the seed oil from homozygous T4 generation seeds revealed that over-expression of the FAE1 gene caused a 36% increase in the percent of erucic acid (37–49% compared to 36% in untransformed control). The down-regulation of FAE1 caused an 86% decrease in the percent of erucic acid to as low as 5% in the seed oil of transgenic plants. Thus, it is clearly possible to alter erucic acid content of mustard by altering the expression level of the FAE 1 gene. S. Kanrar and J. Venkateswari equally contributed to this work.     

Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.)

The quality of canola oil is determined by its constituent fatty acids such as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Most canola cultivars normally produce oil with about 55–65% oleic acid and 8–12% linolenic acid. High concentrations of linolenic acid lead to oil instability and off-type flavor, while high levels of oleic acid increase oxidative stability and nutritional value of oil. Therefore, development of canola cultivars with increased oleic acid and reduced linolenic acid is highly desirable for canola oil quality. In this study, we have mapped one locus that has a major effect and one locus that has a minor effect for high oleic acid and two loci that have major effects for low linolenic acid in a doubled haploid population. The major locus for high C18:1 was proven to be the fatty acid desaturase-2 (fad2) gene and it is located on the linkage group N5; the minor locus is located on N1. One major QTL for C18:3 is the fatty acid desaturase-3 gene of the genome C (fad3c) and it is located on N14. The second major QTL resides on N4 and is the fad3a gene of the A genome. We have sequenced genomic clones of the fad2 and fad3c genes amplified from an EMS-induced mutant and a wild-type canola cultivar. A comparison of the mutant and wild-type allele sequences of the fad2 and fad3c genes revealed single nucleotide mutations in each of the genes. Detailed sequence analyses suggested mechanisms by which both the mutations can cause altered fatty acid content. Based on the sequence differences between the mutant and wild-type alleles, two single nucleotide polymorphism (SNP) markers, corresponding to the fad2 and fad3c gene mutations, were developed. These markers will be highly useful for direct selection of desirable fad2 and fad3c alleles during marker-assisted trait introgression and breeding of canola with high oleic and low linolenic acid.     

芥菜型油菜脂肪酸含量的变异、相关性分析及芥酸调控基因FAE1特异引物设计

芥菜型油菜是我国芸苔属的三大油料作物之一,具有耐旱、抗病虫等优良特性;而我国是芥菜型油菜的重要起源中心,具有丰富的种质资源.该研究从全国各地搜集了34份芥菜型油菜,在贵阳环境条件下种植,其脂肪酸含量(芥酸、油酸、硬脂酸、亚麻酸和亚油酸)表现出丰富的变异,并呈正态分布.结果表明:这些芥菜型油菜种质资源的不同脂肪酸含量间的相关性发现,芥酸和油酸间呈极显著负相关,亚麻酸和硬脂酸呈极显著的正相关,亚麻酸和亚油酸呈现负相关.利用这些材料的脂肪酸含量进行主成分分析,发现绝大部分材料(30份,占88.2%)集中在二维图的特定区域,只有少数其它材料散落在图中其它区域,他们分别是SL63、棱角油菜、T6342和长阳黄芥,这些变异较大的材料在芥菜型油菜的育种中可以发挥特殊作用.此外,运用来自甘蓝型油菜和甘蓝的芥酸调控基因FAE1的已知序列,并设计了FAE1特异引物,而引物在全部34份芥菜型油菜种质资源中均表现出了较好的扩增效果.因此证实芥菜型油菜中至少含有一个FAE1拷贝.该研究结果对于芥菜型油菜育种在我国的开展及其未来的分子育种具有重要的指导意义.     

GLC analysis of Indian rapeseed-mustard to study the variability of fatty acid composition

Rapeseed-mustard is one of the most economically important oilseed crops in India. Speciality oils having high amounts of a specific fatty acid are of immense importance for both nutritional and industrial purposes. Oil high in oleic acid has demand in commercial food-service applications due to a long shelf-life and cholesterol-reducing properties. Both linoleic and linolenic acids are essential fatty acids; however, less than 3% linolenic acid is preferred for oil stability. High erucic acid content is beneficial for the polymer industry, whereas low erucic acid is recommended for food purposes. Therefore, it is important to undertake systematic characterization of the available gene pool for its variable fatty acid profile to be utilized for specific purposes. In the present study the Indian rapeseed-mustard germplasm and some newly developed low-erucic-acid strains were analysed by GLC to study the fatty acid composition in these lines. The GLC analysis revealed that the rapeseed-mustard varieties being commonly grown in India are characterized by high erucic acid content (30-51%) in the oil with low levels of oleic acid (13-23%). However, from among the recently developed low-erucic-acid strains, several lines were identified with comparatively high oleic acid (60-70%), moderate to high linoleic acid (13-40%) and low linolenic acid (< 10%) contents. Work is in progress at TERI (New Delhi, India) to utilize these lines for development of strains with particular fatty acid compositions for specific purposes.     

Mutant alleles of <Emphasis Type="Italic">FAD2-1A</Emphasis> and <Emphasis Type="Italic">FAD2-1B</Emphasis>combine to produce soybeans with the high oleic acid seed oil trait

Background  

The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B.     

Erucic acid metabolism in rat heart. A combined biochemical and radioautographical study.

Metabolism of Erucic Acid was studied in rat heart in comparison with that of oleic acid, particularly in relation with diet lipids. Rats were fed for 3 or 60 days a diet containing 30% of the calories of either Rapessed Oil, rich in erucic acid or sunflower seed oil rich in linoleic acid. They were I.V. injected with tritiated erucic or oleic acid. After 1 or 15 min the radioactivity recovered in heart lipids was very low whatever the diet (1 to 2%). One minute after injection of erucic acid the radioactivity was mainly recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. When oleic was injected, the radioactivity was principally recovered in triacylglycerols as untransformed oleic acid whatever the experimental conditions. Electron microscopy showed that a much higher proportion of peroxisomes, was present in heart cells, following sunflower seed oil diet as compared to rapeseed oil diet. In all cases mitochondria supported the greater part of radioactivity, especially when erucic acid was injected in rats fed rapeseed oil. After sunflower seed oil, a noticeable radioactivity was observed in peroxisomes, most of them containing silver grains, especially when oleic acid was injected. According to the data reported, peroxisomes do not seem more implicated than mitochondria in the metabolism of erucic acid in myocardium.     

Changes in fatty acid composition of lipid classes in developing mustard seed

Maturation of mustard (Sinapis alba) seed proceeds with a sharp decrease in the amounts of palmitic and linoleic acids in the total lipids up to 6 weeks after flowering (WAF). Concomitantly, the concentration of oleic acid increases, reaching a plateau at 4 WAF, which is followed by chain elongation of oleic acid to gadoleic and erucic acids. Compositional changes in constituent fatty acids of individual lipid classes indicate that the very long-chain monounsaturated fatty acids (C₂₀ and C₂₂), as opposed to common long-chain fatty acids (C₁₆ and C₁₈), are metabolized to triacylglycerols mainly by esterification to preformed diacylglycerols and monoacylglycerols, rather than via esterification to glycerol-3-phosphate or lysophosphatidic acids.