Endoplasmic Reticulum-Located PDAT1-2 from Castor Bean Enhances Hydroxy Fatty Acid Accumulation in Transgenic Plants

Abstract

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

“…But in plant species that accumulate exotic fatty acids such as ricinoleic, vernolic, and ␣-eleostearic acid, DGAT2 and PDAT appear to have a more predominant role in TAG synthesis although DGAT1 is also expressed during seed development in these species (20,49,50). In the case of flax, we found gene homologues encoding all major TAG-synthesizing enzymes with the exception of DGAT3.…”

“…A phylogenetic analysis of PDATs including the functionally tested enzymes from Arabidopsis (17) and castor bean (24,50) indicate that LuPDAT1 and RcPDAT1-2 are closely related (supplemental Fig. 2A).…”

Compartmentation of Triacylglycerol Accumulation in Plants

“…Perhaps substrate specificity or specialized isoforms of LPCAT, PDCT, PDAT, and DGAT evolved in castor bean to be specific for hydroxy-FAs generated on PC by the diverged FAD2 (hydroxylase), and this system would be optimized for directing ricinoleic acid into TAG (59). This is consistent with the incremental increases in accumulation of ricinoleic acid in TAG by coexpressing DGAT2 or PDAT from castor bean in conjunction with the FAD2-like hydroxylase in transgenic oilseeds (47,58,59). It is possible that the activity of PDCT or other enzymes from developing castor seed displays specificity for ricinoleoyl-PC, and engineering of these enzymes might further enhance hydroxy-FA accumulation in transgenic plants.…”

“…Phospholipid:DAG acyltransferase (PDAT) was identified and characterized as an acyl-CoA-independent transacylase that synthesizes TAG from PC and DAG, also yielding lyso-PC. PDAT was heralded as a mechanism for channeling unusual FAs into TAG in some plant species due to its markedly distinct comparative substrate specificities (57), and PDAT has been used to enhance the accumulation of TAG with oxygenated FAs in transgenic seeds (58,59). PDAT was shown to participate in TAG biosynthesis in yeast log-phase growth and could partially compensate for loss of DGAT to store TAG during stationary phase (60).…”

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase

“…PUFAs may be directly transferred from PC into TAG via the catalytic action of phospholipid:diacylglycerol acyltransferase (PDAT) (4). Some oilseeds that are high in PUFAs like flax (5) or unusual fatty acids like castor (Ricinus communis) (6,7) appear to have unique PDAT(s) with high selectivity toward these fatty acids. Alternatively, the phosphocholine head group of PUFA-enriched PC may be removed, producing PUFA-enriched diacylglycerol (DAG), which may be further incorporated into TAG via either or both of PDAT and diacylglycerol acyltransferase (DGAT)-catalyzed reactions.…”