Eva Wiberg

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

Abstract. The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80–90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available.

Substrates of diacylglycerol acyltransferase in microsomes from developing oil seeds

Abstract Selectivities for diacylglycerol and acyl-CoA species by oil seed diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) were studied in enzyme assays with microsomal enzymes from developing seeds of castor bean ( Ricinus communis ), Cuphea procumbens and sunflower ( Helianthus annuus ). Seeds of castor bean and Cuphea procumbens accumulate storage lipids with acyl groups that are excluded from membrane lipids (ricinoleoyl and caproyl moieties, respectively), whereas sunflower seeds accumulate only acyl groups that are common for both membrane and storage lipids. Cuphea procumbens DAGAT showed pronounced selectivities for diacylglycerols and acyl-CoAs containing caproyl groups and the castor bean enzyme showed selectivities for substrates containing ricinoleate. These results are in accordance with the suggested role for DAGAT in the selective channelling of the unusual acyl groups into triacylglycerols in these seeds. Cuphea procumbens and sunflower DAGAT showed a selective acylation of acyl-CoA species from mixtures of palmitoyl-, stearoyl- and oleoyl-CoA. Thus, the activity of their DAGATs will also influence the molecular species of ‘common’ triacylglycerol which will be formed.

Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.).

Abstract. The fatty acid composition and content of membrane and storage lipids of two transgenic laurate-producing rape (Brassica napus L.) lines were monitored during seed development. The two lines, the medium-laurate (ML) line and the high-laurate (HL) line, accumulated 34 mol% and 55 mol% of laurate in their seed triacylglycerols, respectively. The diacylglycerols contained about 17 and 33 mol% of laurate in the ML- and HL-lines, respectively, from the mid-stage of seed development up to seed maturity. The ML-line showed a maximal relative laurate content in phosphatidylcholine (17 mol%) at the mid-stage of seed development whereafter the content decreased to 2.7 mol% with seed maturity. The laurate content in phosphatidylcholine was observed to remain high (26 mol%) in the HL-line from the mid-stage to the end of triacylglycerol deposition. Thereafter, the relative content decreased and reached 6.6 mol% in the mature seeds. There was an enhanced activity of lauroyl-phosphatidylcholine- metabolizing enzymes in the seed membranes from laurate-producing lines compared with control lines, which might explain the decrease seen in laurate content in phosphatidylcholine during seed maturation. A comparison of the laurate distribution in the lipids from developing laurate-producing rape seeds and developing seeds from three species naturally accumulating laurate at similar levels revealed differences in laurate metabolism compared with these species. The results suggest that phospholipids and triacylglycerols are synthesized from the same diacylglycerol pool in rape seeds and that rape lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase do not have the same preference for laurate substrates as the corresponding enzymes in seed tissues naturally accumulating this acyl group. In addition, the mechanisms that specifically remove or exclude laurate from membrane lipids appear less effective in rape seed than in tissues naturally evolved to synthesize laurate-rich oils.

Medium chain-length fatty acids in lipids of developing oil palm kernel endosperm

Abstract The content and acyl qulity of triacylglycerol (TAG), diacylglycerol (DAG) and phosphatidylcholine (PC) in developing palm kernels ( Elaeis guineensis ) endosperm were determined between 8–20 weeks after anthesis (WAA). Rapid accumulation of TAG ( ca 65 μmol per kernel per week) started at 13 WAA and continued until 17 WAA. Accumulation of DAG followed a similar pattern to TAG but was two orders of magnitude less in quantity. The content of the major phospholipid, PC, was more or less constant from 8 to 17 WAA. The levels of medium chain-length fatty acids were very low in all lipid classes at 8 WAA. There was a rapid increase in the relative amounts of 12:0 and 14:0 in TAG between 8 and 12 WAA, after which their proportions remained fairly constant throughout kernel development. The content of 12:0 and 14:0 in PC and DAG increased up to 13 WAA, after which there was a substantial decrease. The decrease in the proportions of 12:0 and 14:0 in DAG and PC coincided with the onset of the rapid TAG synthesis. The results are discussed in terms of the regulation of oil biosynthesis in tissues accumulating medium chain-length fatty acids.

Biosynthesis of an Acetylenic Fatty Acid in Microsomal Preparations from Developing Seeds of Crepis Alpina

Over 600 naturally occuring compounds with acetylenic bonds (triple bonds) have been characterized (Bohlmann et al., 1973). Previous in-vivo studies, from mosses accumulating acetylenic fatty acids, indicate that the acetylenic bond is formed by the substraction of two hydrogen atoms from a double bond (Kohn et al., 1994).