Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels

Abstract

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229-241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999-1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.

Figure 1

LPAAT substrate specificity of CLP-expressing canola seeds. A pool of mid-maturation seeds from a control plant (white bars) and from a transgenic plant, pCGN5511-LP004–5 (black bars), were assayed for LPAAT substrate specificity using 12:0-LPA and various ¹⁴C-labeled acyl-CoAs. PA, Phosphatitic acid.

Figure 2

Correlation of 12:0-LPAAT activity with 12:0 accumulation at sn-2. LPAAT activity using 12:0-CoA and 12:0-LPA was determined in membrane fractions derived from developing untransformed, control canola seeds, as well as from independent CLP LP004 transformants. In addition, the proportion of 12:0 at sn-2 was measured in 20-seed pools of mature F₁ seeds derived from crosses of the same set of CLP plants with a homozygous BTE-containing line, DH22. The figure correlates these two determinations. ♦, Control; ×, individual CLP × BTE F₁ seed lots. PA, Phosphatitic acid.

Figure 3

Relationship between total laurate content and trilaurin. Seed oil was extracted from transformed canola plants and the total laurate content of the oil and proportion of trilaurin compared with other TAGs in the oil were determined. Each symbol reflects a sample derived from a seed pool from one dihaploid plant as described. ×, Canola lines transformed with BTE alone; ○, seeds from F₂ plants resulting from crosses of several different Q04 CLP transformants with the homozygous BTE plant DH22. The line was calculated by assuming that laurate was positioned randomly at all three positions of the triglyceride.

Figure 4

Laurate proportion at sn-2 is dependent on total laurate levels and coconut LPAAT. The primary CLP transformants (pCGN5511 in var Quantum) were crossed with the homozygous BTE line DH63. A F₁ plant harboring CLP and BTE alleles was grown. Independently segregating F₂ microspores derived from this plant were made diploid and grown into (homozygous) dihaploid plants as described. The presence or absence of the coconut LPAAT gene in the individual dihaploid plants was determined via PCR of leaf tissue. All plants were selfed, and oil was extracted from the resulting seeds. The sn-2 analysis of seed oil was executed using R. arrhizus lipase. Each symbol represents a seed pool derived from one dihaploid plant. ○, CLP-positive plant; ×, CLP-negative plant. For this analysis, we selected the top 12 laurate producers of the generated dihaploids, as well as randomly chosen plants throughout the laurate range.

Figure 5

Coconut LPAAT can boost laurate levels. Each symbol represents a seed-pool analysis of an individual dihaploid plant. All dihaploid plants resulting from crosses described in Figure 4 were sorted via PCR into a CLP-containing (CLP +) and a CLP-free (CLP −) populations. Plants of both populations were grouped into 1% laurate intervals.

Figure 6

Correlation of oil levels with BTE and CLP. The total seed oil mass as a percentage of dry weight of the dihaploid plants described in Figure 4 were determined by NMR. The data are shown separately for CLP-free (CLP−) and CLP-containing (CLP+) populations, with the oil percentage plotted against total laurate levels.