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  • Soybean Glycine max is an important oilseed and can


    Soybean (Glycine max) is an important oilseed and can be a useful source of epoxy fatty acids (EFAs). This UFA contains an oxygen bridge across adjacent carbon atoms at single or multiple positions in the acyl chain, making them highly reactive and prone to cross-linking, and thus valuable industrial feedstocks (Cahoon and Ohlrogge, 1994, Carlsson, 2009, Dai et al., 2003). Biochemical analysis has shown that the synthesis of EFAs is catalyzed by epoxygenases of either cytochrome P450-type or FAD2-like enzymes in epoxy fatty Ketoprofen accumulating plant seeds (Bafor et al., 1993, Blée, 1998, Cahoon et al., 2002, Lee et al., 1998). 12-epoxyoctadeca-cis-9-enoic acid (Fig. 9) known as vernolic acid (Va) is an 18-carbon Δ12-epoxy fatty acid resulting from the insertion of an oxygen atom at the Δ12 double bond of linoleic acid (C18:2) bound to phosphatidylcholine (PC) in seeds of Euphorbia lagascae and Vernonia galamensis (Bafor et al., 1993, Liu et al., 1998). Epoxygenase genes from V. galamensis, Stokesia laevis (SlEPX) and Crepis palestina had been cloned (Blée, 1998, Hitz, 1998, Lee et al., 1998). Transgenic expression of each of these epoxygenases alone in Arabidopsis can only result in low levels (<10%) of Va (Blée, 1998, Cahoon et al., 2002, Hatanaka and Hildebrand, 2001, Singh et al., 2001). Over-expression of an E. lagascae epoxygenase gene (CYP726A1) led to the accumulation of Δ12-epoxy fatty acid in tobacco (Nicotina tabacum) callus or in somatic soybean embryos at 15% and 8% (w/w) of total fatty acids (Cahoon et al., 2002). Zhou et al. (2006) reported that coexpression of Crepis palaestina Δ12-epoxygenase (Cpal2) with a typical FAD2 gene enhanced Va accumulation up to 21% in a fae1/fad3 Arabidopsis mutant (51% of C18:2) and 17% in cottonseed. Furthermore, it was reported that EFA levels increased up to 30% in the Arabidopsis co-expressing Cpal2 and a DGAT2 from a very high EFA accumulating plant, Bernardia pulchella (Zhou et al., 2008). Developing seed microsomal assays showed that DGATs from V. galamensis and S. laevis have strong substrate preferences for vernolic acid bearing substrates including acyl-CoA and diacylglycerols (DAGs) (Abe et al., 2006). Subsequently, two DGAT1 cDNA clones (namely VgDGAT1a and VgDGAT1b) and a DGAT2 cDNA clone (VgDGAT2) were isolated and characterized from V. galamensis (Yu et al., 2008). Seed-specific expression of SlEPX alone resulted in 5% Va accumulation in the matured transgenic soybean seeds, and coexpression of SlEPX and VgDGAT1a or VgDGAT2 cDNAs, however, increased Va levels up 14.7% and 26.9% in the mature transgenic soybean seeds (Li et al., 2010a). We here show that SlEPX-expression alone also results in additional phenotypic variations, such as altered seed fatty acid profiles, reduced oil accumulation, disordered seed protein storage, decreased seed germination, altered seed size and morphology and impaired growth. More importantly, these disrupted phenotypes caused by over-expression of SlEPX can be complemented by coexpression of VgDGAT1a or VgDGAT2 and this is accompanied by a major increase in the mass of Va accumulating in the seeds. Our results indicate that DGATs in high accumulators of vernolic acids may selectively incorporate Va into TAGs, and consequently alleviate the negative effect of epoxy fatty acids on cellular activities in the transgenic soybeans. The present data provide useful information for further understanding of UFA biosynthesis and optimizations of metabolic engineering designs for sustainable production of various UFAs at high levels in established oilseed crops.
    Material and methods
    Discussion Increasing effort is now focused on engineering oilseeds to provide renewable, cost-competitive and environmentally friendly sources of industrial oils as alternatives to those currently derived mainly from non-renewable petroleum sources. To meet this need, key enzymes responsible for the synthesis of many unusual fatty acids have been identified, and the corresponding genes have also been cloned from several non-agronomic species that can accumulate high levels of those UFAs in their seeds. FAD2-like enzymes can catalyze the synthesis of a remarkable range of industrial-valued UFAs including hydroxy, epoxy and conjugated fatty acids (Cahoon et al., 2007). We previously isolated a S. laevis cDNA (SlEPX) encoding a Δ12-epoxygenase, which converts linoleic acid (C18:2) into vernolic acid in a one step reaction. Seed-specific expression of this gene resulted in accumulation of 2.4% of vernolic acid (Va) in transgenic Arabidopsis seeds (Hatanaka et al., 2004). Seed-specific expression of SlEPX in both soybean somatic embryos and developing seeds also only yielded a small amount of vernolic acid production (Li et al., 2010a), much lower than in S. laevis seeds. When SlEPX was co-expressed with VgDGAT1 or VgDGAT2 in soybean seeds, Va accumulated up to 17% and 25.9% of total soy oil in the double-transgenic soybean (Li et al., 2010a), indicating that DGATs from Va-high accumulators can increase Va accumulation in the SlEPX-transgenic soybean seeds. Here, our data show that expression of SlEPX alone also results in altered phenotypes in soybean seeds, and more importantly, coexpression of VgDGAT1 or VgDGAt2 can alleviate these effects of SlEPX expression on soybean seeds.