Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11184-11188, December 1992 Plant Biology Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco (fatty acid desaturatlon/tranhgenic exp sln/Umbelferae/unsaturated fatty acid) EDGAR B. CAHOON*, JOHN SHANKLINtt, AND JOHN B. OHLROGGE*§ *Department of Botany and Plant Pathology and tDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 Communicated by Paul K. Stumpf, July 8, 1992 ABSTRACT Little is known about the metabolic origin of linic acid from [14C]acyl-ACPs, including (1-14C]18:0ACP (or petroselinic acid (18:1A'b), the principal fatty acid of the seed from [1-14C]18:0-CoA), has yet to be detected in seed extracts oil of most Umbelliferae, Aralaceae, and Garryaceae species. of the Umbelliferae species coriander and carrot (ref. 6 and To eam the possibility that petroselinic acid is the product unpublished data). Lack of a direct assay complicates any ofan acyl-acyl carrier protein (ACP) desaturase, Western blots attempt to characterize the biosynthetic pathway or to purify ofcoriander and other Umbeliferae seed extracts were probed the acyl-ACP desaturase believed to be involved in petrose- with antibodies agains the A9-stearoyl-ACP desaturase of linic acid synthesis. avocado. In these extracts, proteins of 39 and 36 kDa were As an alternative approach, we examined the possibility detected. Of these, only the 36-kDa peptide was specific to that the hypothetical acyl-ACP desaturase associated with tises which synthesize petroselinic acid. A cDNA encoding the petroselinic acid biosynthesis is antigenically related to 36-kDa peptide was isolated from a coriander endosperm A918:0-ACP desaturase. Using antibodies raised against the cDNA library, placed under control of the caulifower mosaic A918:0-ACP desaturase of avocado (11), we have isolated a virus 35S promoter, and Introduced into tobacco by Agroac- cDNA¶ encoding a mature peptide of w36 kDa which is terium lumefaciens-med transformation. Exssn of detected only in tissues that synthesize petroselinic acid. this cDNA in transgenic tobacco callus was accompanied by the Expression of this clone in tobacco, a species that lacks accumulation of petroselinic acid and A4-hexadecenoic acid, petroselinic acid, results in the accumulation ofthis fatty acid both of which were absent from control callus. These results and thus demonstrates that the 36-kDa peptide is a likely demonstrate the involvement of a 36-kDa putative acyl-ACP acyl-ACP desaturase which is sufficient for the production of desaturase in the biosynthetic pathway of petroselinic acid and petroselinic acid in transgenic plant tissue. the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desat- MATERIALS AND METHODS urase. Western Blot Analysis. Plant tissues were homogenized in Petroselinic acid (18:1A5Lis) is an unusual fatty acid that 50 mM potassium phosphate, pH 7.2/2 mM phenylmethane- occurs primarily in seeds of Umbelliferae (or Apiaceae), sulfonyl fluoride/5 mM sodium metabisulfite/5 mM EDTA/5 Araliaceae, and Garryaceae species (1). This fatty acid com- mM isoascorbate (5 ml/g of fresh weight), passed through poses as much as 85% of the total fatty acid of Umbelliferae two layers of Miracloth (Calbiochem), and mixed with SDS/ seeds but is virtually absent from leaves and other tissues of PAGE sample buffer. these plants (1-3). The structure of petroselinic acid differs Protein extracts of transgenic tobacco calli were obtained from that of oleic acid (18:1A9cis), a common plant fatty acid, by homogenization of tissue in 2 ml of 0.7 M sucrose/0.5 M by the position of its double bond. Because of the unsatur- Tris/50 mM EDTA/0.1 M potassium chloride, pH 9.4, con- ation at carbon 6, petroselinic acid is of potential industrial taining 2% (vol/vol) 2-mercaptoethanol and 2 mM phenyl- significance. Through chemical cleavage at its double bond, methanesulfonyl fluoride added just prior to use. The ho- petroselinic acid can be used as a precursor of lauric acid mogenate was mixed thoroughly with 2 ml of phenol and (12:0), which is a component of detergents and surfactants, centrifuged at 3000 x g for 10 min. The upper, phenol phase and adipic acid (6:0 dicarboxylic), which is the monomeric was recovered, and proteins were precipitated with the component of nylon 66. addition of 10 ml of0.1 M ammonium acetate in methanol and The pathway for petroselinic acid biosynthesis has not overnight incubation at -20TC. The protein pellet obtained been previously determined. Monounsaturated fatty acids of following centrifugation was washed sequentially with meth- plants typically derive from the desaturation of C16 and C18 anolic ammonium acetate and acetone, then air-dried prior to saturated fatty acids bound to acyl carrier protein (ACP) or addition of SDS/PAGE sample buffer. to glycerolipids (4, 5). Our preliminary results from a variety Proteins of plant extracts were separated by SDS/PAGE of 14C labeling studies suggest that petroselinic acid is the (12) using 11% (wt/vol) acrylamide gels. Proteins were trans- product of an acyl-ACP desaturase (ref. 6 and unpublished ferred to nitrocellulose and probed with polyclonal, immu- data). The only such enzyme to have been identified in plants noaffinity-purified antibodies raised against the A918:0-ACP is the A9-stearoyl-ACP (A918:0-ACP) desaturase (EC desaturase of avocado as described (11, 13). 1.14.99.6), which catalyzes the conversion of 18:0-ACP to Isolation and Characterization of cDNA Clones. A cDNA 18:1M9-ACP (7-9). This reaction is readily assayable in tissue expression library was prepared using the AZAP II vector extracts of most plants using [14C]18:0-ACP and cofactors (Stratagene) and poly(A)+ RNA isolated from the endosperm including ferredoxin, NADPH, and ferredoxin-NADPH re- ductase (8, 10). However, the in vitro synthesis of petrose- Abbreviations: ACP, acyl carrier protein; 18:0-ACP, stearoyl-ACP. tPresent address: Biology Department, Brookhaven National Lab- oratory, Upton, NY 11973. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" 1The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M93115). 11184 Downloaded by guest on September 27, 2021 Plant Biology: Cahoon et aL Proc. Natl. Acad. Sci. USA 89 (1992) 11185 and embedded embryo of developing seed of coriander of I2 as described (21) except that derivatization was per- (Coriandrum sativum L.) mericarps. The coriander en- formed for 2 hr. Derivatized samples were analyzed by dosperm cDNA library was subjected to immunological GC-MS with a Hewlett-Packard 5890 GC coupled to a screening (14) with antibodies against A918:0-ACP desaturase Hewlett-Packard 5970A mass-selective detector (MSD) us- of avocado (11). Immunopositive clones were purified to ing a 15 m x 0.25 mm i.d. DB-17 column (J&W Scientific, homogeneity, and pBluescript SK(-) phagemid was excised Rancho Cordova, CA) with the oven temperature pro- as described (15). Nucleotide sequence was obtained for both grammed from 1850C to 2300C at 10'C/min. The MSD inlet strands of DNA by dideoxy chain termination using Seque- temperature was 280'C, and the ionizing potential ofthe MSD nase 2.0 (United States Biochemical). A full-length type II was 70 eV. clone was obtained by rescreening the coriander library with a DNA probe derived from a 394-base-pair (bp) Nco I restriction fragment of a partial type II cDNA (14). RESULTS Expression of cDNAs in Escherichia coli. The mature pep- Immunodetection of Two Putative Acyl-ACP Desaturases in tide (native protein minus plastid transit peptide)-encoding Umbelliferae Seed Extracts. A primary goal of our research regions of type I and type II cDNAs were inserted into the was to determine the metabolic origin of the cis-A6 double Nde I site of the pET3a E. coli expression vector (Novagen) bond of petroselinic acid, using seeds of the Umbelliferae (16), following engineering of terminal restriction sites by species coriander and carrot. In preliminary experiments (6), polymerase chain reaction (PCR). PCR primers for the type [1-14C]stearic acid (18:0) or [1-14C]palmitic acid (16:0) fed to I cDNA contained flanking Xba I and Nde I restriction sites endosperm slices of coriander and carrot was incorporated (5' primer, 5'-TGGTCTAGACATATGGCCTCTACTCTTG- into glycerolipids but not desaturated. However, crude ho- GCATC-3'; 3' primer, 5'-ACCTCTAGACATATGTACA- mogenates of coriander endosperm were capable of the de GACCACAATAAA-3'). Type II cDNA primers were de- novo synthesis of petroselinic acid from [2-14C]malonyl-CoA signed with flanking EcoRI and Nde I restriction sites (5' (6). The majority of the resulting [14C]petroselinic acid was primer, 5'-TAGGAATTCATATGGCTTCAACTCTTCAT- identified as free fatty acid, and a smaller portion of the 3'; 3' primer, 5'-ACCGAATTCATATGATGATCTGACG- recovered petroselinic acid was detected in the acyl-ACP 3'). PCR products were ligated into pBluescript KS(+) and pool. These results, therefore, suggested that petroselinic amplified in E. coli prior to insertion in the pET3a vector. acid derived from an acyl-ACP rather than a glycerolipid-type The pET3a-derived plasmids were introduced into E. coli desaturase. Despite this, we were unable to demonstrate the BL21 and grown under carbenicillin selection (16). Cells in vitro synthesis ofpetroselinic acid from [1-14C]stearoyl- or containing expression plasmids lacking insert or with insert [1-14C]palmitoyl-ACP with crude homogenates of coriander encoding type I or type II mature peptide were grown to an or carrot endosperm (unpublished data).