It has been reported that vernolic acid accumulation
It has been reported that vernolic THZ1 Hydrochloride accumulation in transgenic Arabidopsis seeds expressing a Δ12-epoxygenase often resulted in failure of germination and impaired growth and development (Singh et al., 2001). Similar phenomena were also reported for transgenic Arabidopsis seeds with accumulation of hydroxy fatty acids (Browse et al., 2008) and transgenic production of α-eleostearic acid in soybean somatic embryos and in wild-type Arabidopsis seeds by expression of a Momordica charantia conjugase (Cahoon et al., 2006, Cahoon et al., 2001). These observations suggest the possibility that accumulation of unusual fatty acids synthesized by divergent FAD2-like enzymes in heterologous hosts might affect metabolism and physiology of seeds. In our case, a number of detrimental phenotypes were found for SlEPX-expressing soybean seeds. Increased levels of C18:1 was observed in the SlEPX transgenic soybean seeds compared to the controls (Fig. 1). This phenomenon was also observed in other transgenic studies using divergent FAD2-like enzymes operating on different fatty acid substrates and performing a range of different modifications (Broun and Somerville, 1997a, Cahoon et al., 1999, Cahoon et al., 2006, Dyer et al., 2002, Lee et al., 1998, Singh et al., 2001, Smith et al., 2003, van de Loo et al., 1995, Zhou et al., 2006). One of the bases for this variation might be a homology-dependent interaction between endogenous FAD2 and transgenically expressed FAD2-like enzymes, which may suppress the endogenous FAD2 activity (Singh et al., 2001). It is proposed that the UFA products in some way may inhibit the activity of the endogenous FAD2 enzyme thus blocking the conversion of oleic acid to linoleic acid (Broun and Somerville, 1997b, Cahoon et al., 2002). Two major altered phenotypes were reductions in total oil contents and large variations in protein levels for SlEPX-expressing soybean seeds (Fig. 2, Fig. 3). Some reports suggest that UFAs such as hydroxy fatty acids produced in the transgenic plants are targeted for degradation by β-oxidation (Moire et al., 2004). β-oxidation may result in specific breakdown of the UFAs, leading to a low level of the target UFAs in the transgenic seeds (Broun et al., 1998, Eccleston and Ohlrogge, 1998). This might cause a futile cycle of the synthesis and degradation of other normal FA thereby reducting total oil content (Eccleston and Ohlrogge, 1998). Another possibility is that the unusual Δ12-modified fatty acids such as Va may not be efficiently removed from phosphatidylcholine (PC) (in membrane lipids) after synthesis (Fig. 9). Further, the UFA accumulation on PC might affect the functions of the other enzymes associated with FA and TAG biosynthesis pathway. Also, the accumulation of PC molecules bearing Va might account for the large variation in protein content although the mechanism underlying this phenotype in the SlEPX-transgenic soybean seeds remains to be addressed. Interestingly and importantly, coexpression of VgDGAT1 and VgDGAT2 in the SlEPX-transgenic lines reversed the reduction in seed oil content and protein levels were restored to normal in the soybean seeds (Fig. 2, Fig. 3). The increase in total oil was due to increases in Va and restoration of other common FAs except for decreased C18:2 (Li et al., 2010a), suggesting that increased rates of β-oxidation may breakdown both common and UFAs such as Va. C18:2 reductions can be explained by its conversion to Va. A previous report demonstrated increased rates of non-selective FA β-oxidation in mutant plants deficient in accumulation of palmitic acid (Bonaventure et al., 2004). We hypothesize that an increased rate of Va sequestration in TAG by VgDGAT1 and VgDGAT2 activity may prevent increased rates of total FA β-oxidation. This may reduce futile cycling of FA synthesis and degradation, which could recover the total oil content. Alternatively feedback inhibition of FA synthesis might also be involved in reducing FA accumulation. A build up of acyl-CoA has been shown to down regulate FA synthesis (Shintani and Ohlrogge, 1995). When soybean acyltransferases do not efficiently utilize Va-CoA then their build up may cause feedback inhibition of FA synthesis. VgDGAT1/2 can efficiently utilize Va-CoA to produce Va-TAG which may limit accumulation of Va-CoA (Fig. 9). If so, this may lower the amount of Va-CoA and alleviate the reduced TAG accumulation. Further analysis of this phenomenon may identify new engineering strategies for accumulation of unusual FAs in oilseeds.