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Functional Plant Biology, 2005, 32, 473–479

www.publish.csiro.au/journals/fpb

Rapid communication: Metabolic engineering of Arabidopsis to produce nutritionally important DHA in seed oil Stan S. RobertA,B,D , Surinder P. SinghA,C,D , Xue-Rong ZhouA,C,D , James R. PetrieA,C , Susan I. BlackburnA,B , Peter M. MansourA,B , Peter D. NicholsA,B , Qing LiuA,C and Allan G. GreenA,C,E A Food

Futures National Research Flagship. Marine Research, GPO Box 1538, Hobart, Tas. 7001, Australia. C CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia. D These authors contributed equally. E Corresponding author. Email: [email protected]

B CSIRO

Abstract. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are nutritionally important long-chain (≥ C20 ) omega-3 polyunsaturated fatty acids (ω3 LC-PUFA) currently obtained mainly from marine sources. A set of genes encoding the fatty acid chain elongation and desaturation enzymes required for the synthesis of LCPUFA from their C18 PUFA precursors was expressed seed-specifically in Arabidopsis thaliana. This resulted in the synthesis of DHA, the most nutritionally important ω3 LC-PUFA, for the first time in seed oils, along with its precursor EPA and the ω6 LC-PUFA arachidonic acid (ARA). The assembled pathway utilised 5 and 6 desaturases that operate on acyl-CoA substrates and led to higher levels of synthesis of LC-PUFA than previously reported with acyl-PC desaturases. This demonstrates the potential for development of land plants as alternative sources of DHA and other LC-PUFA to meet the growing demand for these nutrients. Keywords: desaturase, DHA, elongase, EPA, genetic engineering, omega-3 fatty acid, seed oil.

Introduction Increased levels of docosahexaenoic acid (DHA, 22 : 64,7,10,13,16,19 ) and other omega-3 long-chain polyunsaturated fatty acids (ω3 LC-PUFA) in the human diet have been consistently associated with a broad range of health benefits, including improved brain and retinal development, and reduced risk of coronary heart disease and type II diabetes (Simopoulos 2003). Although the human body is capable of synthesising DHA from dietary α-linolenic acid (ALA, 18 : 39,12,15 ), the conversion rate is very inefficient, particularly in the case of modern diets rich in linoleic acid (LA, 18 : 29,12 ) an ω6-PUFA that competes with ALA for the enzymes involved in DHA synthesis (Voss et al. 1991). Therefore, various health authorities now recommend increased dietary intake of DHA and other ω3 LC-PUFA (Simopoulos 2003). The current main dietary

source of ω3 LC-PUFA is fish and other seafood, which actually acquire these LC-PUFA in their diets from lower marine plant forms such as microalgae and thraustochytrids. However, the irrefutable weight of evidence now shows that global wild fisheries are fully exploited and many are on the verge of collapse (Myers and Worm 2003). In addition, many aquaculture systems rely heavily on wild fisheries for feeds, and are net consumers, not producers, of ω3 LC-PUFA. This combination of factors could lead to serious consequences for both human health and marine resource sustainability unless alternative sources of ω3 LC-PUFA can be found (Nichols 2004). Plant oils have the potential to be developed into sustainable and affordable sources of ω3 LC-PUFA through the transgenic expression of genes encoding LC-PUFA biosynthetic pathways from other organisms

Abbreviations used: ALA, α-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; GC, gas chromatography; GC–MS, GC mass spectroscopy; GLA, γ -linolenic acid; hph, hygromycin B phosphotransferase gene; LA, linoleic acid; LC-PUFA, long-chain polyunsaturated fatty acids; nos, nopaline synthase; NptII, neomycin phosphotransferase II gene; SDA, stearidonic acid; TAG, triacylglycerol; X : Y, a fatty acid containing X carbons with Y double bonds. © CSIRO 2005

10.1071/FP05084

1445-4408/05/060473

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(Singh et al. 2005). All higher plants have the ability to synthesise the main C18 -PUFA, LA and ALA, and a limited number can also synthesise γ -linolenic acid (GLA, 18 : 36,9,12 ) and stearidonic acid (SDA, 18 : 46,9,12,15 ). However, they universally lack the genetic capability to further elongate and desaturate these C18 -PUFA to produce LC-PUFA. Genes encoding the additional enzymes required for synthesis of LC-PUFA from C18 -PUFA in plants can potentially be obtained from a range of LC-PUFA-synthesising organisms, such as microalgae, thraustochytrids, fungi, mosses, and bacteria. These organisms synthesise LC-PUFA by a variety of routes including both aerobic (desaturase and elongase) and anaerobic (polyketide synthase) pathways (Sayanova and Napier 2004). The aerobic pathway consists of consecutive cycles of chain elongation and desaturation. Pathway variations exist for the order in which the desaturation and elongation steps leading to C20 -PUFA can occur (Fig. 1). The ‘6 pathway’ involves sequential 6-desaturation, chain elongation, and 5-desaturation, to yield eicosapentaenoic acid (EPA, 20 : 55,8,11,14,17 ) when ALA is the initial substrate (Fig. 1A) and arachidonic acid (ARA, 20 : 45,8,11,14 ) when LA is the initial substrate. The alternative ‘8 pathway’ commences instead with

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the C18 to C20 chain elongation step, followed by consecutive 8-desaturation and 5-desaturation (Fig. 1B). In both pathways, EPA is subsequently elongated to docosapentaenoic acid (22 : 57,10,13,16,19 , DPA) and then desaturated at the 4 position to yield DHA. Previous attempts to engineer C20 -PUFA synthesis by introducing either the 8 pathway in Arabidopsis leaves (Qi et al. 2004) or the 6 pathway in linseed (Abbadi et al. 2004) resulted in only low levels of EPA and ARA. The low C20 -PUFA synthesis was attributed to inefficient transfer of the fatty acid substrates and intermediates between the acyl-PC pool, where desaturation occurs, and the acylCoA pool, where chain elongation occurs (Abbadi et al. 2004; Singh et al. 2005). Here we describe the transgenic expression of an alternative LC-PUFA metabolic pathway (Fig. 1C) devised to avoid this impediment by utilising a dual-purpose 5 / 6-desaturase that acts on acyl-CoA substrates rather than the acyl-PC substrates utilised by the individual 6, 5 and 8-desaturases (Fig. 1A, B) expressed in previous studies (Abbadi et al. 2004; Qi et al. 2004). This approach avoids the need for substrate-switching between the acyl-PC and acyl-CoA pools, a process mediated by acyltransferases with appropriate LC-PUFA specificities that could be lacking in plants.

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Fig. 1. Alternative pathways for synthesis of the ω3 LC-PUFA SDA (18 : 4), EPA (20 : 5) and DHA (22 : 6) from ALA (18 : 3). Desaturases, elongases and acyltransferases are shown as solid, open and dashed arrows respectively. Chain elongation occurs only on acyl-CoA substrates, whereas desaturation can occur on either acyl-PC (A, B) or acyl-CoA substrates (C). The acyl-PC or acyl-CoA substrate preference of the final 4-desaturase step has not yet been determined. Pathways involving acyl-PC desaturases require acyltransferase-mediated shuttling of acyl groups between the PC and CoA substrates. Panels A and B show the ‘6 pathway’ and ‘8 pathway’ variants of the acyl-PC desaturase pathway respectively. Panel C shows the pathway expressed in the current study in which the acyl-CoA 6 and 5-desaturase activities were encoded by the zebrafish 5 / 6 dual-function desaturase. Synthesis of ω6 LC-PUFA such as ARA (20 : 4) occurs by the same set of reactions but commencing with LA (18 : 2) as the initial substrate.

Metabolic engineering of Arabidopsis to produce DHA in seed oil

Functional Plant Biology

We demonstrate, for the first time, the synthesis in seeds of all three nutritionally important LC-PUFA, namely ARA, EPA and DHA. We chose Arabidopsis thaliana as the model plant to express the LC-PUFA synthesis pathway because its seed oil contains significant amounts of the LA and ALA precursors required for ω6 and ω3 LC-PUFA synthesis respectively. Materials and methods Genes and constructs The full LC-PUFA pathway was introduced into Arabidopsis thaliana (L.) Heynh. by sequential transformation with two separate binary vectors containing first an ‘EPA construct’ which encoded the enzymes required for conversion of ALA to EPA (and LA to ARA) and second a ‘DHA construct’ encoding the additional enzymes required to convert EPA to DHA (Fig. 2). The EPA construct consisted of the zebrafish (Danio rerio) 5 / 6desaturase gene (Accession number AF309556) and the nematode (Caenorhabditis elegans) 6-elongase gene (Accession number Z68749). Each gene was placed under the independent control of a seed-specific napin promoter, designated Fp1 (Stalberg et al. 1993). For plant transformation, the genes were inserted into the binary vector pWVec8 that contained a spectinomycin resistance gene for selection in E. coli and an enhanced hygromycin resistance gene (hph) for selection in plants (Wang et al. 1997). To achieve this, the C. elegans 6-elongase gene coding region was PCR amplified with primers, sense 5 -GCGGGTACCATGGCTCAGCATCCGCTC-3 and antisense 5 -GCGGGATCCTTAGTTGTTCTTCTTCTT-3 , and inserted as a blunt-end fragment between the Fp1 and nos 3 terminator fragments in a binary vector pWVec8 derivative, forming pCeloPWVec8. The zebrafish 5 / 6-desaturase gene coding region was initially PCR amplified with primers, sense 5 -CCCAAGCTTACTATGGGTGGCGGAGGACA GC-3 and antisense 5 -CCGCTGGAGTTATTTGTTGAGATACGC-3 ,

and inserted as a blunt end fragment between the Fp1 and nos 3 terminator sequences in a pBluescript SK (Stratagene) derivative. Subsequently, the entire vector containing the desaturase expression cassette was inserted into the HindIII site of pCeloPWVec8, forming pSSP-5 / 6D.6E. Taq polymerase was used in both amplifications and the resulting amplified fragments were sequenced to confirm the absence of any PCR-induced errors. The DHA construct consisted of the microalga Pavlova salina 4-desaturase and the 5-elongase genes (Accession numbers AY926605 and AY926606, respectively). The coding region of the 5-elongase gene was excised from its cDNA clone in pBluescript SK (Stratagene) as a PstI–SacII fragment and inserted into an intermediate plasmid pXZP143 between the napin promoter Fp1 and nos terminator in pBluescript SK backbone, resulting in plasmid pXZP144. The coding region of the 4-desaturase gene was excised from its cDNA clone in pBluescript SK as a BamHI–SalI fragment and inserted into plasmid pXZP143 between the napin promoter Fp1 and nos terminator, resulting in plasmid pXZP150. These two expression cassettes were combined in one vector by inserting the HindIII–ApaI fragment from pXZP144 (containing Fp1-5Elo-nos3 ) between the StuI and ApaI sites of pXZP150 immediate downstream of Fp1-4Des-nos3 expression cassette, resulting in plasmid pXZP191. The HindIII–StuI fragment from pXZP191 containing the two pyramided expression cassettes was then cloned into the binary vector pBI121, with kanamycin resistance gene as a selectable marker for selection in plant cells and in E. coli, resulting in plant expression vector pXZP355. Both EPA and DHA constructs are shown schematically in Fig. 2, and were introduced into Agrobacterium strain AGL1 (Valvekens et al. 1988) by electroporation before plant transformation. Plant materials and transformation Arabidopsis thaliana (ecotype Columbia) was used for all plant transformations. Agrobacterium transformation was carried out by the floral dipping method (Clough and Bent 1998). T1 seeds harvested from the treated plants were plated out on selective media containing

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Fig. 2. Transgene constructs used to express genes encoding LC-PUFA biosynthetic enzymes in Arabidopsis. The EPA construct consisted of the zebrafish dual function 5 / 6-desaturase (D5 / D6Des) and the nematode 6-elongase (D6Elo) both driven by the truncated napin promoter (Fp1). The DHA construct consisted of the Pavlova salina 4-desaturase (D4Des) and 5-elongase (D5Elo) genes both driven by the truncated napin promoter (Fp1).

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hygromycin (20 mg L−1 ) or kanamycin (40 mg L−1 ) as appropriate, and transformed plants selected and transferred to soil to establish T1 plants.

A 18:1ω9

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Oil extraction, lipid class and fatty acid analyses Total seed oil was obtained by a one-phase chloroform : methanol : water extraction method (Bligh and Dyer 1959). Fractionation of seed oil was performed by silicic acid column chromatography (Leblond and Chapman 2000) with neutral lipid (predominately triacylglycerol), glycolipid, and phospholipids being eluted respectively with chloroform, acetone, and 0.1% glacial acetic acid in methanol. The lipid class distribution in the seeds was determined by Iatroscan TLC–FID analysis (Volkman and Nichols 1991) of an aliquot of the total seed oil. Fatty acid compositions were determined by GC and GC–MS analysis (Bransden et al. 2005) of fatty acid methyl esters prepared by direct transmethylation of either the whole seeds or the separated triacylglycerol and phospholipid fractions obtained from total seed oil (Fig. 3).

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Following transformation with the EPA construct and selection on hygromycin, 26 T1 plants were recovered and their seed analysed for fatty acid composition. Twenty-five of these T1 plants showed synthesis of new ω3 PUFA, including SDA, 20 : 48,11,14,17 and EPA, and new ω6 PUFA, including GLA, 20 : 38,11,14 and ARA. EPA levels ranged from 0.4% to 2.3% and ARA from 0.2% to 1.4% across the 25 T1 plants. One of the highest EPA-containing lines (DO11, 2.3% EPA) was confirmed to be a single locus transformation event by the 3 : 1 segregation of the hygromycin-resistance gene in its T2 seed. A T2 progeny plant (DO11-5) derived from DO11 and verified as homozygous for the EPA construct synthesised a total of 9.6% new ω3 and ω6 PUFA, including 3.2% EPA and 1.6% ARA (Table 1). Unexpectedly, DO11-5 also contained 0.1% of DPA, the elongation product of EPA. This reveals that the nematode 6-elongase also has some 5-elongase capability when expressed in plants, although no such activity was found previously when expressed in yeast (Napier and Michaelson 2001). The level of EPA synthesis achieved in Arabidopsis seeds is 4-fold higher than the 0.8% level previously attained in linseed (Abbadi et al. 2004). Considering also that the level of ALA precursor for EPA synthesis in Arabidopsis seed is less than a third of that present in linseed, it appears that the acyl-CoA desaturasemediated pathway for C18 and C20 PUFA synthesis expressed in our study is operating with significantly greater efficiency than the acyl-PC desaturase pathway expressed in linseed. Prior to the derivation of homozygotes from the DO11 plant, a T2 seedling population segregating for the EPA construct was transformed with the DHA construct using the same methods as for the EPA construct. T1 plants were established from seedlings recovered following germination on combined hygromycin and kanamycin media. Fatty acid analysis of selfed seed showed significant conversion of EPA to DHA had occurred in two T1 plants, with DW2 and DW5 having 0.2% and 0.5% DHA respectively (Table 1). The

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Results and discussion

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Fig. 3. (A) Gas chromatogram showing fatty acid profile for Arabidopsis line DW5 carrying EPA and DHA gene constructs (upper panel) compared to wild-type Arabidopsis (lower panel). (B) Mass spectra for EPA and DHA obtained from Arabidopsis thaliana line DW5.

presence of both the EPA and DHA constructs in DW2 and DW5 was further confirmed by successful PCR amplification of each of the four component desaturase and elongase genes. Germination of 50 T2 seeds from each of DW2 and DW5 on hygromycin-containing medium showed that the DW5 T1 plant was homozygous for the EPA construct, whereas

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Table 1. Fatty acid composition (% of total fatty acids) of total seed lipids from Arabidopsis thaliana (Columbia) and derivatives carrying EPA and DHA gene constructs Fatty acid composition of triacylglycerol (TAG) and phospholipid (PL) fractions of DW5 are also shown Columbia wild type

Columbia + EPA construct DO11–5

7.2 2.9 20.0 27.5 15.1 2.2 19.8 2.2 0.1 1.5 1.5 100.0

7.3 4.3 19.1 26.4 11.7 1.8 11.1 2.7 3.5 0.8 1.7 90.4

6.7 3.8 20.6 26.0 13.2 2.1 14.8 3.0 1.7 1.4 2.9 96.0

6.1 4.4 16.6 25.9 15.0 1.8 10.5 4.2 3.5 1.0 2.7 91.7

5.5 4.3 18.9 25.5 13.6 1.9 10.5 4.8 3.8 0.3 2.4 91.5

12.5 4.5 13.7 33.1 15.1 0.6 3.2 1.4 3.7 0.4 3.8 92.0

New ω6 PUFA 18 : 36,9,12 (GLA) 20 : 38,11,14 20 : 45,8,11,14 (ARA) 22 : 47,10,13,16 22 : 54,7,10,13,16 Total

0 0 0 0 0 0

0.6 1.9 1.6 0 0 4.1

0.2 0.8 0.4 0 0 1.4

0.4 1.5 1.0 0 0.1 3.0

0.4 1.5 1.1 0 0.1 3.1

0.2 1.7 1.2 0.2 0.1 3.4

New ω3 PUFA 18 : 46,9,12,15 (SDA) 20 : 48,11,14,17 20 : 55,8,11,14,17 (EPA) 22 : 57,10,13,16,19 (DPA) 22 : 64,7,10,13,16,19 (DHA) Total

0 0 0 0 0 0

1.8 0.4 3.2 0.1 0 5.5

0.7 0.5 1.1 0.1 0.2 2.6

1.5 0.8 2.4 0.1 0.5 5.3

1.6 0.7 2.5 0.2 0.4 5.4

0.5 0.9 2.3 0.7 0.2 4.6

100.0 41.3 42.6 0

100.0 31.0 38.1 9.6

100.0 36.8 39.2 4.0

100.0 28.1 40.9 8.3

100.0 29.7 39.1 8.5

100.0 17.3 48.2 8.0

Fatty acid Usual fatty acids 16 : 0 18 : 0 18 : 19 18 : 29,12 (LA) 18 : 39,12,15 (ALA) 20 : 0 20 : 111 20 : 113 20 : 211,14 22 : 113 Other minor Total

Total fatty acids Total MUFAA Total C18 -PUFAB Total new PUFAC

DO11 + DHA construct DW2 DW5 DW5 DW5 Total Total TAG PL

Total of 18 : 19 and derived LC-MUFA (= 18 : 19 + 20 : 111 + 22 : 113 ). 18 : 2 + 18 : 3. C Total of all new ω6 and ω3-PUFA. A B

DW2 segregated in a 3 : 1 ratio (resistant : susceptible) and was therefore heterozygous for the EPA construct. This was consistent with the higher levels of EPA observed in DW5 seed compared to DW2 seed, and explained the higher level of DHA produced in DW5. In addition, as the DHA construct is segregating in the seed borne on these T1 plants, the pooled fatty acid composition represents an average of a range of null, heterozygous and homozygous genotypes for the DHA construct. Thus, it is possible that levels of DHA will rise in transgene homozygotes eventually isolated from the progeny of DW5. Fractionation of the total seed lipids of DW5 seed revealed them to be comprised of 89% TAG and 11% polar lipids (largely made up phospholipids). Furthermore, fatty acid analysis of the TAG fraction from

DW5 seed showed that the newly synthesised EPA and DHA were being incorporated into the seed oil and that the proportion of EPA and DHA in the fatty acid composition of the total seed lipid essentially reflected that of the TAG fraction (Table 1). The relative efficiencies of the individual enzymatic steps encoded by the EPA construct can be assessed by examining the percentage conversion of substrate fatty acid to product fatty acids (including subsequent derivatives) in DO11-5. The zebrafish 5 / 6 desaturase exhibited only moderate 6-desaturation activity with only 32% of ALA and 14% LA being converted to 6-desaturated PUFA products. However, it showed strong 5-desaturation,

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with 89% of 20 : 4ω3 being converted to EPA and DPA, and 45% of 20 : 3ω6 being converted to ARA. Given that previous studies in yeast showed this enzyme to actually have higher 6-desaturase activity than 5-desaturase activity, the relatively low 6-desaturation levels achieved in Arabidopsis seeds could indicate a limited availability of ALA and LA substrates in the acyl-CoA pool (Singh et al. 2005). The preference of the zebrafish 5 / 6 desaturase for ω3-PUFA over ω6-PUFA substrates is consistent with that previously reported for this enzyme when expressed in yeast (Hastings et al. 2001). The nematode 6-elongase operated highly efficiently, with 86% of GLA and 67% of SDA being elongated, suggesting that this enzyme may have a slight preference for elongation of ω6-PUFA substrate. Examination of the enzyme efficiencies in the highest DHA plant (DW5) shows that only 17% of EPA was elongated to DPA by the P. salina 5-elongase, whereas virtually all of this DPA was converted to DHA by the P. salina 4-desaturase. It is interesting to also note the consequences of LCPUFA synthesis on the overall fatty acid profile. Although more than 8% of new ω6 and ω3 PUFA were synthesised in both DO11–5 and DW5, there was relatively little reduction in the levels of respective LA and ALA precursors (Table 1). Instead, the level of monounsaturated 18 : 19 and its elongated derivatives (20 : 111 and 22 : 113 ) declined significantly. Thus it appears that conversion of C18 -PUFA to LC-PUFA results in increased conversion of 18 : 1 to LA and ALA, and a corresponding reduction in 18 : 1 available for elongation. This is the first report of successful transgenic assembly of a complete and effective DHA biosynthetic pathway in seeds. The initial concentrations of DHA and other LCPUFA in these seed oils are modest in comparison with those in fish, microalgae and other microorganisms. However, there is considerable potential for increased levels to be achieved with the acyl-CoA desaturase pathway approach utilised in this study, such as through the introduction of more efficient forms of the enzymes obtained from other source organisms, enhancement of expression levels of the introduced genes by alternative promoters, and the introduction of appropriate acyltransferases to raise C18 -PUFA levels in the acyl-CoA pool. Importantly, the overall levels of LC-PUFA synthesis should be significantly higher when these pathways are introduced into those oilseeds that contain higher levels of available LA and ALA substrates in their seed oils than are present in Arabidopsis. The development of DHA-containing seeds provides a powerful example of the ability to incorporate complex metabolic pathways into plants to enhance their nutritional value. With considerable direct nutritional benefits to consumers, and the potential to reduce pressure on depleted global fish stocks, plant products containing

S. S. Robert et al.

EPA and DHA are expected to have substantial appeal to consumers. Acknowledgments This research was conducted as part of the Food Futures Flagship, one of CSIRO’s National Research Flagships. We acknowledge the expert technical assistance provided by Mina Brock, Samantha Chhe, Dion Frampton, Diana Hall, Clive Hurlstone, Bronwyn Innes and Adam White. References Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. The Plant Cell 16, 2734–2748. doi: 10.1105/tpc.104.026070 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. Bransden MP, Battaglene SC, Dunstan GA, Morehead DT, Nichols PD (2005) Effect of dietary 22 : 6n-3 on growth, survival and tissue fatty acid profile of striped trumpeter (Latris lineata) larvae fed enriched Artemia. Aquaculture 243, 331–344. doi: 10.1016/j.aquaculture.2004.11.002 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743. doi: 10.1046/j.1365313x.1998.00343.x Hastings N, Agaba M, Tocher DR, Leaver MJ, Dick JR, Sargent JR, Teale AJ (2001) A vertebrate fatty acid desaturase with 5 and 6 activities. Proceedings of the National Academy of Sciences USA 98, 14304–14309. doi: 10.1073/ pnas.251516598 Leblond JD, Chapman PJ (2000) Lipid class distribution of highly unsaturated long chain fatty acids in marine dinoflagellates. Journal of Phycology 36, 1103–1108. doi: 10.1046/j.15298817.2000.00018.x Myers RA, Worm B (2003) Rapid worldwide depletion of predatory fish communities. Nature 423, 280–283. doi: 10.1038/ nature01610 Napier JA, Michaelson LV (2001) Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans. Lipids 36, 761–766. Nichols P (2004) Sources of long-chain omega-3 oils. Lipid Technology 16, 247–251. Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier JA, Stobart AK, Lazarus CM (2004) Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nature Biotechnology 22, 739–745. doi: 10.1038/nbt972 Sayanova OV, Napier JA (2004) Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants. Phytochemistry 65, 147–158. doi: 10.1016/ j.phytochem.2003.10.017 Simopoulos AP (2003) Importance of the ratio of omega-6 / omega-3 essential fatty acids: evolutionary aspects. World Review of Nutrition and Dietetics 92, 1–22. Singh SP, Zhou X-R, Liu Q, Stymne S, Green AG (2005) Metabolic engineering of new fatty acids in plants. Current Opinion in Plant Biology 8, 197–203. doi: 10.1016/j.pbi.2005.01.012

Metabolic engineering of Arabidopsis to produce DHA in seed oil

Stalberg K, Ellerstrom M, Josefsson LG, Rask L (1993) Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco. Plant Molecular Biology 23, 671–683. doi: 10.1007/BF00021523 Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proceedings of the National Academy of Sciences USA 85, 5536–5540. Volkman JK, Nichols PD (1991) Applications of thin-layer chromatography-flame ionization detection to the analysis of lipids and pollutants in marine and environmental samples. Journal of Planar Chromatography – Modern TLC 4, 19–26.

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Voss A, Reinhart M, Sankarappa S, Sprecher H (1991) The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase. Journal of Biological Chemistry 266, 19995–20000. Wang MB, Upadhyaya NM, Brettell RIS, Waterhouse PM (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. Journal of Genetics and Breeding 51, 325–334.

Manuscript received 14 April 2005, accepted 29 April 2005

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