The utilization of the acyl-CoA and the involvement PDAT and DGAT in the biosynthesis of erucic acid-rich triacylglycerols in Crambe seed oil

Abstract

The triacylglycerol of Crambe abyssinica seeds consist of 95% very long chain (>18 carbon) fatty acids (86% erucic acid; 22:1∆13) in the sn-1 and sn-3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10% of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl-CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0-CoA and 18:1-CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl-CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl-CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl-CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl-CoA by the acyltransferases in the glycerol-3-phosphate pathway.

Scheme 1

Proposed schematic flow of oleoyl (18:1) and erucoyl (22:1) acyl groups to triacylglycerols (TAG) in Crambe seed cells. The thickness of the filled arrows represents roughly the proportions of the various flows. Oleate produced in the plastid is exported out in the cytosol where enzymes in the endoplasmic reticulum catalyze the depicted reactions. Oleate from oleoyl-CoA enters phosphatidylcholine (PdtCho) reversibly via the acyl-CoA:lysophosphatidylcholine (LPCAT) catalyzed reaction. The main part of the oleoyl-CoA is elongated to erucoyl-CoA which is used by the acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) in the formation of lysophosphatidic acid (lysoPtdtOH) and the acyl-CoA:diacylglycerol acyltransferase (DGAT) in the formation of triacylglycerols. The oleoyl-CoA and other C18 acyl groups are esterified mainly at the sn-2 position of the glycerol backbone to form phosphatidic acid (PtdOH). Triacylglycerol formation also occurs via the phospholipid:diacylglycerol acyltransferase (PDAT). The DGAT catalyzed reaction results in TAG with mainly erucic acid in the outer positions and C18 fatty acids in the middle, sn-2 position whereas the (minor) PDAT catalyzed reaction results in TAG with erucic acid only at the sn-1 position

Fig. 1

Total fatty acids and erucic acid (nmol/seed) in developing Crambe seeds at different days after flowering (DAF)

Fig. 2

Erucoyl-CoA content in developing Crambe seeds at different days after flowering (DAF). a Amount of total acyl-CoA and erucoyl-CoA in Crambe seeds. b The percentage of erucoyl groups in acyl-CoA and complex lipids in Crambe seeds

Fig. 3

Phospholipid:diacylglycerol acyltransferase (PDAT) and acyl-CoA:diacylglycerol acyltransferase (DGAT) activities in microsomal preparations from developing Crambe seeds at different days after flowering (DAF). DGAT activity was measured with different acyl-CoAs as indicated in the figure

Fig. 4

Ratio of specific activity in the acylation of added di-6:0-diacylglyceols (DAG) to endogenous DAG by membrane preparations prepared from Crambe seeds at different days after flowering (DAF). DGAT activity was measured with different acyl-CoAs as indicated in the figure