As shown in Fig there are two mechanisms

As shown in Fig. 9, there are two mechanisms for the removal of the Va-acyl group from PC to make it available for incorporation into TAG with DGATs\' acting at the final acylation step. ①: Transfer of Va from PC to the acyl-CoA pool. This process can be driven by the reverse action of acyl- CoA:lysophosphatidylcholine acyltransferase (LPCAT) (Stymne and Stobart, 1984), or the combined action of phospholipase A2 (PLA2) and a long chain acyl-CoA synthetase (LACS). Va in the acyl-CoA pool can be transfer to glycerol-3-phosphate (G3P) by acyl-CoA:G3P acyltransferase (GPAT) forming lysophosphatic filipin (LPA) by acyl-CoA:lysophosphatidic acid acyltransferase (LPAT), form diacylglycerol (DAG) phosphatidic acid phosphatase (PAP), and finally be incorporated into TAG by acyl-CoA:diacylglycerol acyltransferase (DGAT) through the Kennedy pathway involved in Brown et al. (2002), Lassner et al. (1995), and Katavic et al. (1995)). ②: Removal of the PC phosphocholine headgroup to produce diacylglycerol (DAG) containing Va of the PC. This reaction can proceed by several enzymatic actions, such as the reverse action of CDP-choline:diacylglycerol cholinephosphotransferase (CPT) (Slack et al., 1983), or phosphatidylcholine: diacyglycerol cholinephosphotransferase (PDCT) recently identified in Arabidopsis (Lu et al., 2009). This process can also involve phospholipase C, phospholipase D along with PAP (van Erp et al., 2011). For our case, the Va-DAG formed by these enzymatic mechanisms can be substrates for transfer of Va from Va-CoA by DGATs producing Va-TAG. Our previous data showed that microsomal VgDGATs have higher specificity for acyl substrates containing Va (Yu et al., 2006). Here, analysis of lipid classes in transgenic soybean seeds and Stokesia and Vernonia seeds demonstrated that the Va-PC level is higher while Va-TAG is lower in the SlEPX-transgenic seeds. However, Va accumulation in PC is low in both the double-transgenic soybean seeds and Stokesia and Vernonia seeds where most of Va are in TAG (Fig. 8). The reduction of Va in PC by VgDGATs presumably occurs indirectly as a result of removal of Va from the acyl-CoA and DAG pools. FAs esterified to PC are under a constant dynamic exchange with the acyl-CoA and DAG pools (Fig. 9), involving a cycle of deacylation and reacylation of PC (Bates et al., 2009, Bates et al., 2007, Lu et al., 2009, Napier and Graham, 2010). Therefore, Va-CoAs that are not rapidly utilized by soybean acyltransferases, may be reincorporated into PC through the acyl editing cycle. Removal of Va-CoAs from the acyl-CoA and DAG pools may cause a net flux of Va out of PC into TAG (Fig. 9). This indicates that most Va bound to PC produced by SlEPX was removed and subsequently incorporated into TAG by VgDGATs in the double-transgenic soybean seeds the same as in Va-high accumulators. Accompanying the newly synthesized Va-PC transfer to TAG, all disordered phenotypes in SlEPX-expressing soybeans were restored to normal. Other recent studies have also shown the need for enhancement of multiple steps of metabolic pathways for high accumulation of desired products in transgenic plants and other organisms. Sugiyama et al. (2011) for example found that over-expression of prenyltransferase genes in the legume, Lotus japonicus, leads to production of prenylated polyphenols only when flavonoid substrates are supplied. Dulermo and Nicaud (2011) were able to drive TAG accumulation to high levels in the oleaginous yeast Yarrowia lipolytica by the synergistic expression of acyltransferase to G3P synthesis genes along with reduced β-oxidation. Algal experiments by Li et al. (2010b) show that TAG levels can increase 10-fold by reducing hydrocarbon flow into starch by down regulating ADP-glucose pyrophosphorylase. To greatly increase TAG levels in maize embryos metabolic flux analyses indicate that redirecting hydrocarbon flow into TAG will be limited by NADPH demands for fatty acid biosynthesis (Paula Alonso et al., 2010).