Phosphatidylcholine:diacylglycerol cholinephosphotransferase's unique regulation of castor bean oil quality

Abstract

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with "common" acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

Figure 1

Utilization of de novo synthesized DAG with and without ricinoleoyl groups by Camelina PDCT. Formation of radioactive lipids from [¹⁴C] G3P, 18:3-CoA (A) and ricinoleoyl-CoA (Ric-CoA) (B) as single acyl-CoA substrate or in mixtures in proportions 2:3 (C) in microsomal preparations of H1246 yeast expressing Camelina PDCT. 0-OH-DAG, 1-OH-DAG, and 2-OH-DAG, DAGs with no, one, and two ricinoleoyl groups, respectively; 0-OH-PC, 1-OH-PC and 2-OH-PC, PC with no, one, and two ricinoleyl groups, respectively. Average value shown ± sd, n = 3 replicates.

Figure 2

Utilization of de novo synthesized DAG with and without ricinoleoyl groups by castor PDCT. Formation of radioactive lipids from [¹⁴C]G3P, 18:3-CoA (A) and ricinoleoyl-CoA (Ric-CoA) (B), presented as single substrate or in mixtures in proportions 2:3 (C). Activities were measured in microsomal preparations of H1246 yeast expressing castor PDCT. 0-OH-DAG, 1-OH-DAG and 2-OH-DAG, DAG, with one, two and three ricinoleoyl groups, respectively; 0-OH-PC, 1-OH-PC, PC with no, one and two ricinoleoyl groups, respectively. Average value shown ± sd, n = 3 replicates.

Figure 3

Accumulation of radioactive lipids from [¹⁴C]G3P and a mixture of 18:1-CoA and ricinoleoyl-CoA in microsomal preparation of developing castor endosperm. Incubations were performed for 30 and 60 min as indicated in the figure and contained 150 µg of castor microsomal protein, 12.5 nmol of [¹⁴C]G3P, 12.5 nmol 18:1-CoA, and 12.5 nmol of ricinoleoyl-CoA in total volume of 100 µL of 0.1 M Tris–HCl buffer, pH 7.2. A, Amount of radioactivity lipids in DAG, TAG, and PC. B, Amount of radioactivity in PA. 0-OH-DAG, 1-OH-DAG and 2-OH-DAG, DAGs with no, one and two ricinoleoyl groups, respectively. 0-OH-PC, 1-OH-PC and 2-OH-PC, PC with no, one and two ricinoleoyl groups. 0-OH-TAG, 1-OH-TAG, 2-OH-TAG and 3-OH-TAG, TAG with no, one, two and three ricinoleoyl groups, respectively. Average value shown ± sd, n = 3 replicates.

Figure 4

Acyl-CoA specificity of castor DGAT2 with different DAG species. Acyl-CoA specificity of castor DGAT2 was assessed with DAG with two ricinoleoyl groups (2-OH-DAG) (A), one ricinoleoyl group (1-OH-DAG) (B), di-18:1-DAG (C), and di-18:2-DAG (D) as acyl acceptors. The DGAT2 activity was measured in microsomal preparations of yeast strain H1246 expressing the castor DGAT2. Average values shown ± sd, n = 3 replicates. Double (**) asterisks indicate significant difference at P < 0.01 in one-way ANOVA followed by post hoc Tukey’s test.

Figure 5

Selectivity for DAG species by castor DGAT2 with ricinoleoyl-CoA as acyl donor. Castor DGAT2 selectivity for DAG species was assessed with [¹⁴C]ricinoleoyl-CoA as acyl donor together with an equimolar mixture of sn-1,2-DAG composed of di-18:1-DAG, 1-OH-DAG and 2-OH-DAG (A) and di-18:2-DAG, 1-OH-DAG, and 2-OH-DAG (B) as acyl acceptors. The DGAT2 activity was measured in microsomal preparations of yeast strain H1246 expressing the castor DGAT2. 1-OH-TAG, 2-OH-TAG, and 3-OH-TAG designate TAG with no, one, two, and three ricinoleoyl groups. Average values shown ± sd, n = 3 replicates. Single (*) or double (**) asterisks indicate significant difference at P < 0.05 or at P < 0.01, respectively, in one-way ANOVA followed by post hoc Tukey’s test.

Figure 6

Separation and quantification of DAG species in microsomal preparations of castor bean endosperm. A, TLC separation of DAG species in microsomal preparation of developing castor endosperm visualized in UV-light after sprayed with primuline. Areas containing the separated DAG species are encircled with pencil and were removed for analysis of acyl composition and quantity. B, The amounts of separated DAG species from (A) as determined by GC. The relative contribution (%) of each DAG species is indicated in the figure. 0-OH-DAG, 1-OH-DAG, and 2 OH-DAG designate DAG with no, one and two ricinoleoyl groups respectively. Average values shown ± sd, n = 3 replicates.

Figure 7

Acyl composition of PC and two TLC separated 0-OH-DAG bands in microsomal preparations from developing castor bean endosperm. The PC species and 0-OH-DAG species were separated in two different TLC system, in situ methylated, and the acyl composition determined by GC. The two separated 0-OH-DAG bands were from TLC plate presented in Figure 6A. 0-OH-DAG designate DAG with no ricinoleoyl group. Average values shown ± sd, n = 3 replicates.

Figure 8

Positional distribution of nonhydroxylated fatty acids in 2-OH-TAG from castor oil and the nondiscrimative nature of the TAG lipase. A, Lipid products from 2-OH-TAG from castor oil treated with lipase from R. miehei. B, Lipid products from an equimolar mixture of tri-18:1-TAG and 3-OH-TAG treated with lipase from R. miehei The 2-OH-TAG and 3-OH-TAG were first isolated from castor oil by TLC, eluted and quantified by GC before used for lipase assays. 0-OH, 1-OH, 2-OH, and 3-OH indicate the absence of ricinoleoyl groups, 1 ricinoleoyl group, 2 ricinoleoyl groups, and 3 ricinoleoyl groups, respectively, in TAG, DAG, and MAG. Average values shown ± sd, n = 3 replicates from the same lipase-treated sample.

Figure 9

Distribution of [¹⁴C]18:1 in PC and LPC after acylation with added sn-1-18:1-LPC or sn-1-ricinoleoyl-LPC in microsomal preparations from developing castor bean endosperm. Castor microsomal preparations (150 µg of microsomal protein) were incubated with 15 nmol of either sn-1-18:1-LPC or 15 nmol sn-1-ricinoleoyl-LPC (ric-LPC) and 12 nmol of [¹⁴C]18:1-CoA as indicated in the figure for 30 min. 0-OH-PC and 1-OH-PC designate phsophatidylcholine with no and one ricinoleoyl group, respectively; LPC, lysophosphatidylcholine. Average values shown ± sd, n = 3 replicates.

Figure 10

Castor PDCT and DGAT2 working in concert to enhance tri-ricinoleoyl glycerol amount in castor seeds. Proposed acyl flow in TAG synthetic pathway in castor endosperm. De novo synthesis of DAG via G3P pathway gives rise to DAG with two (2-OH-DAG) and one (1-OH-DAG) ricinoleoyl groups (mainly located in the sn-2 position). 1-OH-DAG is preferentially exchanged with PC with no ricinoleoyl groups (0-OH-PC) by PDCT. The ricinoleoyl group in 1-OH-PC derived from DAG is rapidly replaced with a nonhydroxylated acyl group and the ricinoleate is recycled in the G3P pathway to mainly yield 2-OH-DAG or used by PDAT to form 3-OH-TAG. Ricinoleoyl-containing DAG is mainly acylated with ricinoleoyl groups by DGAT2. However, DAG with no ricinoleoyl groups (0-OH-DAG) is strongly selected against in presence of DAG with ricinoleoyl groups. Thus, a DAG pool with acyl composition similar to PC and not used in TAG synthesis replaces the 1-OH-DAG shunted to PC and can be used in membrane lipid synthesis. PDAT reaction is not depicted in the figure but can be predicted to have the same high selectivity for ricinoleoyl-containing substrates as DGAT2 (Dahlqvist et al., 2000; Lunn et al. 2019). ER, endoplasmic reticulum.