Anders Dahlqvist

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower ( Carthamus tinctorius L.) seeds

Abstract. Microsomal membrane preparations from the immature cotyledons of safflower (Carthamus tinctorius) catalysed the interconversion of the neutral lipids, mono-, di-, and triacylglycerol. Membranes were incubated with neutral lipid substrates, 14C-labelled either in the acyl or glycerol moiety, and the incorporation of radioactivity into other complex lipids determined. It was clear that diacylglycerol gave rise to triacylglycerol and monoacylglycerol as well as phosphatidylcholine. Radioactivity from added [14C] triacylglycerol was to a small extent transferred to diacylglycerol whereas added [14C] monoacylglycerol was rapidly converted to diacylglycerols and triacylglycerols. The formation of triacylglycerol from diacylglycerol occurred in the absence of acyl-CoA and hence did not involve diacylglycerol acyltransferase (DAGAT) activity. Monoacylglycerol was not esterified by direct acylation from acyl-CoA. We propose that these reactions were catalyzed by a diacylglycerol: diacylglycerol transacylase which yielded triacylglycerol and monoacylglycerol, the reaction being freely reversible. The specific activity of the transacylase was some 25% of the diacylglycerol acyltransferase activity and, hence, during the net accumulation of oil, substantial newly formed triacylglycerol equilibrated with the diacylglycerol pool. In its turn the diacylglycerol rapidly interconverted with phosphatidylcholine, the major complex lipid substrate for Δ12 desaturation. Hence, the oleate from triacylglycerols entering phosphatidylcholine via this route could be further desaturated to linoleate. A model is presented which reconciles these observations with our current understanding of fatty acid desaturation in phosphatidylcholine and oil assembly in oleaceous seeds.