Abstract
Petroselinic acid (18:1Δ6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the Δ4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of Δ4-hexadecenoyl (16:1Δ4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1Δ4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1Δ4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1Δ4-ACP. In addition, the elongation of 16:1Δ4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 μM. This finding suggested that the elongation of 16:1Δ4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) I-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I, its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.
Similar content being viewed by others
References
Bradford, M.M. 1976. A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Cahoon, E.B. and Ohlrogge, J.B. 1994a. Metabolic evidence for the involvement of a Δ4-palmitoyl-acyl carrier protein desaturase in petroselinic acid synthesis in coriander endosperm and transgenic tobacco cells. Plant Physiol. 104: 827–837.
Cahoon, E.B. and Ohlrogge, J.B. 1994b. Apparent role of phosphatidylcholine in the metabolism of petroselinic acid in developing Umbelliferae endosperm. Plant Physiol. 104: 845–855.
Cahoon, E.B., Shanklin, J. and Ohlrogge, J.B. 1992. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc. Natl. Acad. Sci. USA 89: 11184–11188.
Christie, W.W. 1982. Lipid Analysis, 2nd ed. Pergamon Press, Oxford.
Crowe, J.S., Cooper, H.J., Smith, M.A., Sims, M.J., Parker, D. and Gewert, D. 1991. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucl. Acids Res. 19: 184.
Dehesh, K., Edwards, P., Fillatti, J., Slabaugh, M. and Byrne, J. 1998. KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme. Plant J. 15: 383–390.
Dörmann, P., Frentzen, M. and Ohlrogge, J.B. 1994. Specificities of acyl-acyl carrier protein (ACP) thioesterase and glycerol-3-phosphate acyltransferase for octadecenoyl-ACP isomers. Identification of a petroselinoyl-ACP thioesterase in Umbelliferae. Plant Physiol. 104: 839–844.
Dutta, P.C., and Appelqvist, L.-A. 1991. Lipids and fatty acid patterns in developing seed, leaf, root, and in tissue culture initiated from embryos of Daucus carota L. Plant Sci. 75: 177–183.
Emanuelson, O., Nielsen, H. and von Heijne, G. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8: 978–984.
Frohman, M.A. 1990. RACE: rapid amplification of cDNA ends. In: M.A., Innis, D.H. Gelfand, J.J. Sninsky and T.J. White, (Eds.) PCR protocols, Academic Press, New York.
Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY.
Jaworski, J., Clough, R. and Barnum, S. 1989. A cerulenin insensitive short chain 3-ketoacyl-acyl carrier protein synthase in Spinacia oleracea leaves. Plant Physiol. 90: 41–44.
Kleiman, R. and Spencer, G.F. 1982. Search for new industrial oils.: XVI. Umbelliferae-seed oils rich in petroselinic acid. J. Am. Oil. Chem. Soc. 59: 29–38.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227: 680–685.
Lee, C., Levin, A. and Branton, D. 1987. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal. Biochem. 166: 308–312.
Leonard, J.M., Knapp, S.J. and Slabaugh, M.B. 1998. A Cuphea ß-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases. Plant J. 13: 621–628.
Magnuson, K., Jackowski, S., Rock, C.O. and Cronan, J.E. 1993. Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol. Rev. 57: 522–542.
Millar, A.A. and Kunst, L. 1995. Isolation of an Arabidopsis cDNA encoding 3-ketoacyl-acyl carrier protein synthase I. Plant Physiol. 108: 1747.
Morris, M.A., Wharry, D.M. and Hammond, E.W. 1967. Chro-matographic behaviour of isomeric long-chain aliphatic com-pounds. II. Argentation thin-layer chromatography of isomeric octadecanoates. J. Chromatog. 31: 69–76.
Morrison, W.R. and Smith, L.M. 1964. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron trifluoride methanol. J. Lipid. Res. 5: 600–608.
Page, R.D.M. 1996. TREEVIEW: an application to display phylo-genetic trees on personal computers. Comp. Appl. Biosci. 12: 357–358.
Rock, C.O. and Garwin, J.L. 1979. Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J. Biol. Chem. 254: 7123–7128.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning:. A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Schultz, D.J., Suh, M.C. and Ohlrogge, J.B. 2000. Stearoyl-acyl carrier protein and unusualacyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin. Plant Physiol. 124: 681–692.
Shimakata, T. and Stumpf, P.K. 1982. Isolation and function of spinach leaf β-ketoacyl-[acyl-carrier-protein] synthases. Proc. Natl. Acad. Sci. USA 79: 5808–5812.
Slabaugh, M.B., Leonard, J.M. and Knapp, S.J. 1998. Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis. Plant J. 13: 611–620.
Suh, M.C., Schultz, D.J. and Ohlrogge, J.B. 1999. Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis. Plant J. 17: 679–688.
Towbin, E., Staehelin, T. and Gordon, J. 1979. Electrophoretic trans-fer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.
Wissenbach, M. 1994. New members of the barley Kas gene fam-ily encoding ß-ketoacyl-acyl carrier protein synthases. Plant Physiol. 106: 1711–1712.
Wu, J., James D.W. Jr., Dooner, H.K. and Browse, J. 1994. A mutant of Arabidopsis deficient in the elongation of palmitic acid. Plant Physiol. 106: 143–150.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mekhedov, S., Cahoon, E.B. & Ohlrogge, J. An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander. Plant Mol Biol 47, 507–518 (2001). https://doi.org/10.1023/A:1011832611885
Issue Date:
DOI: https://doi.org/10.1023/A:1011832611885