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Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

ABSTRACT: The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.

SUBMITTER: Smith MA

PROVIDER: S-EPMC1149651 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Project description:Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].

Project description:BACKGROUND: The application and nutritional value of vegetable oil is highly dependent on its fatty acid composition, especially the relative proportion of its two major fatty acids, i.e oleic acid and linoleic acid. Microsomal oleoyl phosphatidylcholine desaturase encoded by FAD2 gene is known to introduce a double bond at the ?12 position of an oleic acid on phosphatidylcholine and convert it to linoleic acid. The known plant FAD2 enzymes are encoded by small gene families consisting of 1-4 members. In addition to the classic oleate ?12-desaturation activity, functional variants of FAD2 that are capable of undertaking additional or alternative acyl modifications have also been reported in a limited number of plant species. In this study, our objective was to identify FAD2 genes from safflower and analyse their differential expression profile and potentially diversified functionality. RESULTS: We report here the characterization and functional expression of an exceptionally large FAD2 gene family from safflower, and the temporal and spatial expression profiles of these genes as revealed through Real-Time quantitative PCR. The diversified functionalities of some of the safflower FAD2 gene family members were demonstrated by ectopic expression in yeast and transient expression in Nicotiana benthamiana leaves. CtFAD2-1 and CtFAD2-10 were demonstrated to be oleate desaturases specifically expressed in developing seeds and flower head, respectively, while CtFAD2-2 appears to have relatively low oleate desaturation activity throughout the plant. CtFAD2-5 and CtFAD2-8 are specifically expressed in root tissues, while CtFAD2-3, 4, 6, 7 are mostly expressed in the cotyledons and hypocotyls in young safflower seedlings. CtFAD2-9 was found to encode a novel desaturase operating on C16:1 substrate. CtFAD2-11 is a tri-functional enzyme able to introduce a carbon double bond in either cis or trans configuration, or a carbon triple (acetylenic) bond at the ?12 position. CONCLUSIONS: In this study, we isolated an unusually large FAD2 gene family with 11 members from safflower. The seed expressed FAD2 oleate ?12 desaturase genes identified in this study will provide candidate targets to manipulate the oleic acid level in safflower seed oil. Further, the divergent FAD2 enzymes with novel functionality could be used to produce rare fatty acids, such as crepenynic acid, in genetically engineered crop plants that are precursors for economically important phytoalexins and oleochemical products.

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Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

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