Synthesis of trilaurin by developing pisa seeds (Actinodaphne hookeri).

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C16:0, C18:0, C18:2, and C18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% PL. At this stage the TAG contained exclusively C12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C12:0. Experiments on [1-14C]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in PL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into PL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into PL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering. Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C12:0 and 61% of C14:0, but not C16:0, C18:2, and C18:3, into TAG. In contrast the LCFA were incorporated preferentially into PL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering.