Abstract
We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.
Highlights
We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis
These results indicated that only about half of the hydroxy fatty acids (HFAs) were incorporated into TAG in the rod1 mutant compared with the wild type and that this inefficiency was presumably caused by the loss of the PDCT function in the rod1 mutant
These results demonstrated that RcROD1 was able to significantly increase HFA accumulation when coexpressed with the castor FAH12 in Arabidopsis seeds, and this increase was stably inherited over multiple generations
Summary
We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. In Arabidopsis (Arabidopsis thaliana) seeds, 18:1-CoA may be elongated into 20:1-CoA to 22:1-CoA by a fatty acid elongase (FAE1) on the ER membrane (Kunst et al, 1992; Kunst and Samuels, 2003) or may be incorporated into the membrane lipid phosphatidylcholine (PC) to be desaturated to linoleic acid (18:2) and subsequently to linolenic acid (18:3) by the oleate desaturase FAD2 and the linoleate desaturase FAD3, located on the ER (Browse et al, 1993; Sperling et al, 1993; Okuley et al, 1994) These reactions are the major causes of the wide variations of FA composition in TAG of many oilseeds.
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