Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11184-11188, December 1992 Biology Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco ( 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 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the Umbelliferae species coriander and carrot (ref. 6 and To eam the possibility that petroselinic acid is the product unpublished data).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 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 ), sulfonyl fluoride/5 mM sodium metabisulfite/5 mM EDTA/5 , 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 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 (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 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 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] (18:0) or [1-14C] (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). OD6w of 0.6, induced with isopropyl 3-D-thiogalactopyran- Using an alternative approach, we investigated the possi- oside (0.4 mM) for 4 hr, and pelleted. Proteins extracted from bility that the proposed acyl-ACP desaturase involved in these cells were separated by SDS/PAGE and analyzed by petroselinic acid synthesis is related to the A918:0-ACP Coomassie staining or by Western blotting as above. In the desaturase, the only known acyl-ACP desaturase in plants. case of BL21 cells with type I-containing plasmids, washed This enzyme has been purified to homogeneity from avocado pelleted cells were lysed by four cycles of freezing in liquid (11), safflower (22), and soybean (23, 24), and corresponding nitrogen followed by thawing in a 25°C water bath. Cell debris cDNAs were isolated from castor (11), cucumber (11), saf- was pelleted and the soluble extract was used for A918:0-ACP flower (22), and soybean (23). Antibodies raised against the desaturase assay (10). A918:0-ACP desaturase of avocado (11) were used to probe Expression of Type II cDNA in Tobacco. The full-length Western blots of extracts of developing seeds of coriander coriander type II cDNA was inserted behind the cauliflower and several other Umbelliferae species. In these extracts, mosaic virus 35S promoter in the BamHI and Sac I sites of two immunoreactive proteins of apparent mass of 39 and 36 the plant expression vector pBI121 (Clontech) (17). The kDa were detected (Fig. 1A, lanes C-F). In contrast, tissues insert was generated from a full-length type II cDNA by PCR that do not synthesize petroselinic acid-e.g., leaves and and included 5' and 3' restriction sites (5' primer, 5'- roots of coriander (Fig. 1B) and seeds of species outside of TAGGATCCATGGCCATGAAACTGAAT-3'; 3' primer, 5'- the Umbelliferae, Araliaceae, and Garryaceae families (Fig. ACGGATCCGAGCTCTCGACGACCACTCATATG-3'). 1A, lane B)-contained only a 39-kDa protein with antigenic The PCR product was ligated into the BamHI site of pBlue- recognition by anti-A918:0-ACP desaturase antibodies. From script and amplified in E. coli prior to insertion in pBI121. The these results, it was hypothesized that the 39-kDa peptide resulting pBI121-derived plasmid was introduced into Agro- was a A918:0-ACP desaturase, and the additional 36-kDa bacterium tumefaciens LBA4404 by electroporation (18). peptide, seen only in tissues which contain petroselinic acid, The transformed Agrobacterium cells were grown at 27°C was an acyl-ACP desaturase associated with the synthesis of under kanamycin selection and cocultivated with leafdisks of this fatty acid. tobacco (Nicotiana tabacum L.) as described (19). Isolation of Putative Coriander Acyl-ACP Desaturase c- Fatty Acid Analysis of Transgenic Tobacco Callus. Fatty DNAs. As an initial step in determining the function ofthe 39- acid methyl were prepared from transgenic tobacco and 36-kDa peptides identified on Western blots, clones calli by heating of tissue at 90°C for 40 min in 10%o (wt/vol) encoding acyl-ACP desaturases were isolated from a cDNA boron trichloride/methanol (Alltech) supplemented with 15% expression library prepared from coriander endosperm. toluene (vol/vol) (13). Resulting methyl esters were analyzed Screening of the library with antibodies against avocado by gas chromatography using a 50 m x 0.25 mm i.d. CP-Sil88 A918:0-ACP desaturase yielded two classes of clones, desig- column (Chrompack) and oven temperature programming nated type I and type II, as determined by partial nucleotide from 155°C (60-min hold) to 175°C at 2.5°C/min with a column sequencing. Translation of the 5' nucleotide sequence re- head pressure of 7.5 psi (1 psi = 6.89 kPa) of helium. vealed that both type I and type II cDNAs contained con- Monounsaturated fatty acid methyl esters for analysis by siderable amino acid sequence similarity with those of pre- gas chromatography-mass spectroscopy (GC-MS) were sep- viously isolated A918:0-ACP desaturase cDNAs of castor arated by argentation TLC (20) and recovered from TLC (11), cucumber (11), and safflower (22). However, a region plates as described (13). Double bonds of purified monoun- near the amino termini of translated type II clones exhibited saturated fatty acids were converted to thiomethyl adducts marked divergence with a similar region in translated type I by reaction with dimethyl disulfide (Aldrich) in the presence (data not shown) and A918:0-ACP desaturase (Fig. 2) clones. Downloaded by guest on September 27, 2021 11186 Plant Biology: Cahoon et al. Proc. Nad. Acad. Sci. USA 89 (199-2) A, J) Gurr:ifer;ao Urmibellferae quences of both cDNAs were engineered into the pET3a :L.) A B3£ C D E F vector and expressed in E. coli BL21 (16). Proteins encoded ..,.": by the type I and type II cDNAs were readily detectable in Coomassie-stained SDS/polyacrylamide gels ofcrude E. coli protein extracts (data not shown). These proteins also dis- played immunological cross-reactivity with anti-A918:0-ACP desaturase antibodies. In addition, the SDS/PAGE mobility 43 of the type I-encoded peptide was identical to that of the m 441,,1o.u -.lm 14mlm < 3 kv 39-kDa protein detected in coriander seed extracts (data not -_ __ . _ :-3; -La, in vitro 2 shown) and possessed A918:0-ACP desaturase activ- -ok~~~~~~~~~~~~~~~~~~~~~~~~~~I u5r. ity (10) (data not shown). The type I cDNA was therefore identified as encoding a 39-kDa A918:0-ACP desaturase. Western blots of extracts of E. coli expressing the type II clone revealed a 36-kDa immunoreactive peptide indistin- guishable from the lower band of coriander seed extracts (data not shown). The type II-encoded peptide, however, was B 1L.r. 4 V c-r:: detected almost entirely as an insoluble aggregate in E. coli extracts. As a result, we were unable to assay for activity directly, and preliminary attempts to obtain in vitro activity 4 :. 36 r, D of the type II-encoded peptide after urea solubilization of protein aggregates were unsuccessful. Expression of the Coriander Type II cDNA in Tobacco. To FIG. 1. Western blots of seed extracts of Cruciferae and Umbel- directly test the hypothesis that the 36-kDa peptide is an liferae spp. (A) and , root, and seed extracts of coriander (B) acyl-ACP desaturase that results in petroselinic acid accu- probed with anti-A918:0-ACP desaturase antibodies. (A) Lanes: A, mulation, the type II cDNA was cloned into the plant castor recombinant A918:0-ACP desaturase (Ds; 600 ng; see ref. 11); expression vector pBI121 (17) and transformed into tobacco, B, crambe (Crambe abyssinica L.); C, coriander; D, wild carrot a no (Daucus carota L.); E, sweet cicely (Myrrhis odorata L.); F, plant which contains detectable petroselinic acid. Prob- angelica (Angelica archangelica L.). Fifty to 60 ,ug of seed extract ing of Western blots of transgenic tobacco callus with anti- protein was loaded in lanes B-F. Lane B is a Cruciferae species. A918:0-ACP desaturase antibodies revealed an additional Lanes C-F are Umbelliferae species. (B): Leaf (110 j.g), root (110 peptide in extracts ofcallus containing the type II cDNA that ,ug), and seed (60 ,ug) extracts of coriander. was absent from callus transformed with only the pBI121 vector (Fig. 3). This peptide possessed a similar mobility in This divergence included the absence of a sequence encoding SDS/PAGE as the 36-kDa peptide in coriander seed extracts. 15 amino acids in the type II clone relative to the type I and To determine whether the peptide encoded by the type II to the previously reported A918:0-ACP desaturase cDNAs cDNA possessed in vivo activity, the fatty acid composition (11, 22). ofthe transgenic callus was examined by gas chromatography The A918:0-ACP desaturase is found in plastids of plant (Fig. 4). Callus transformed with the type II cDNA contained cells (5). A similar localization would be expected for other two fatty acids not present in callus transformed with the acyl-ACP desaturases. However, the longest type I and II pBI121 vector alone (Fig. 4B, peaks 2 and 5). The retention cDNA clones obtained through antibody screening of the times of methyl derivatives of these fatty acids coin- expression library lacked nucleotide sequence coding for an cided with those of methyl hexadecenoic acid (16:1) and entire plastid transit peptide. A full-length type II clone (Fig. methyl petroselinic acid. In 10 callus samples analyzed, 2) was isolated by screening the coriander cDNA library with levels of each of these fatty acids ranged from 1% to 4% a nucleotide probe derived from a 394-bp Nco I restriction (wt/wt) of the total. To determine the position of the double fragment of a partial type II cDNA. The full-length 1309-bp bond ofthe hexadecenoic acid and confirm the position ofthe cDNA clone contained a methionine codon at nucleotide 7 double bond ofpetroselinic acid, monounsaturated fatty acid with surrounding bases that differed by only one nucleotide methyl esters of the transgenic calli were derivatized with from a plant consensus translational start site (25). The open dimethyl disulfide (21) and analyzed by GC-MS (Fig. 5). The reading frame of the type II cDNA consisted of 1155 nucle- mass spectrum of the derivatized petroselinic acid contained otides encoding a 385-amino acid peptide of 43.8 kDa. By the same diagnostic ions as a petroselinic acid standard. In homology with the deduced amino acid sequences of A918:0- addition, the mass spectrum of the derivatized hexadecenoic ACP desaturase cDNAs (11, 22), the coding sequence of the acid of transgenic calli contained ions diagnostic for a A4 mature protein most likely begins at nucleotide 115 of the isomer (27). type II cDNA. The 108 bases preceding the start of the mature peptide encoded a 36-amino acid sequence with DISCUSSION properties consistent with those of a plastid transit peptide (26). The transit peptide encoded by the type II cDNA was Previous metabolic studies have suggested that petroselinic 3 amino acids longer and possessed a distinctly different acid arises from the activity of an acyl-ACP desaturase in amino acid sequence than those encoded by the A918:0-ACP seed of the Umbelliferae species carrot and coriander (ref. 6 desaturase cDNAs reported to date (11, 22). In addition to the and unpublished data). Consistent with this, we have iden- absence of 15 amino acids near its amino terminus, the type tified a 36-kDa peptide that displays immunological cross- II cDNA-encoded peptide differed from that of A918:0-ACP reactivity with an antibody against the A918:0-ACP desatu- desaturase (11, 22) by the presence of one additional amino rase of avocado and is detectable only in tissues that syn- acid near its carboxyl terminus. Excluding missing amino thesize petroselinic acid. Expression of a coriander cDNA acids, the overall amino acid sequence identity ofthe mature encoding this peptide results in the accumulation of petrose- type II-encoded peptide and the mature castor A918:0-ACP linic acid in transgenic callus oftobacco, a plant that does not desaturase (11) was 70%. normally contain this fatty acid. These results provide strong Expression of Type I and Type II Coriander cDNAs in E. evidence that the biosynthetic pathway of petroselinic acid coli. In an attempt to establish the identity of the type I and involves a desaturation step catalyzed by a 36-kDa peptide type II coriander clones, the mature peptide-encoding se- that is antigenically related to the A918:0-ACP desaturase. Downloaded by guest on September 27, 2021 Plant Biology: Cahoon et al. Proc. Nati. Acad. Sci. USA 89 (1992) 11187

TIII CAATGCTAATATCCCTATTCGGCAAAGAAGTTCAATGCCCTAGAGAATAACAGTTCTGT 120 TII M A N K L N A L M T L Q C P K R N N F T R I A P P Q A GR V R S K V S N A S 38 CAS N A L KN P F L S Q T Q K L P 5 F A L N S T R S PK F Y 35 TI I ACTCTTCATGCTAGCCCACTGGTGTTCGACAAGCTGAAGGCTGGGAGGCCT...... GAGGTG...... GATGAATTGTTCAACTCT 195 TII T L H A SP L V F D KL K A G R P - - - E V.------D EL F N S 63 CAS K S G S K E V E N K P F N PP R H V Q V T H S N P P Q K I I K 75 TI I CTGGGTGCAGAACTCTTCCTAACGAAACCTGACOAGCACGCGTCAACGTCTTAGTAGCAG 315 TiI L E G W A R D N I L V H L K S V E N S N 0 P 0 D Y L P D P T S D A F E D 0 V K E 103 CAS D N E E P K C : F A G D E R 115 TI I ATAAACGCAGAACCGTATCTGTTCTTGAAAGTATAGGCCCCATAAGCAGTACGTTAGCTA 435 TII N R E R A K D I P D E Y F V V L V G D N I T E E A L P T Y N S N L N R C D G I K 143 CAS L E D .. : :: : : : : Q T T L V R 155 TIII AGCCGCCCACATCTGCATGACGGTGATCGGGGACCAGCACTTACATTTTTTTTGCATGT 555 TiI D D T G A Q P T S W A T N T R AN T A EE N RH G D LLN K Y L Y L S G R V D N 183 195 675 TII R N I E K T IQ Y L I G S G N D T K T E N C P Y N G F I Y T S F Q K R A T F I S 213 235 CAS G R 0 K E H I K I T A: E 795 253 275 TII 915 TII L A ElI D P D T T V I A F S D NNMR K K I M P A HA M Y D G S D D M L F K H F 303 CAS F : G L A : S L R N D 315 TI I CGCTGTACGTGATTCCGAGGATCGGCTATATTTGGAAAGACGTCAGTAAGCGCGTAGGGAG1035 TII T A V A 0 0 I G V Y S A W DY C D I I D F L V D K W N V A K N T G L S G E G R K 343 CAS S R L T K A : L E : : G RK DL : A Q : 355 TIII CCAATTTTTGTGCGTAACGAATGGAAGTCAGCAGGAAACGGTCTTGTTACGATTACGCG 1155 TII A Q EY V CS L A A K I R R V E E K V 0 G K E K K A V L P V A F S N I F N R 0 I 383 CAS : D R P P R L R A :: R A : E: P T - N P D V 394 TIII TAAGGGTGCAATATTAATTCATTCTTCTTCTTGTTATTTTTCTTCACGCGGTGTTAACG 1275 TII I I * 385 CAS K L * 396 1309 FIG. 2. Nucleotide sequence ofthe coriander type II acyl-ACPdesaturase cDNA (TiI) and a comparison ofthe deduced amino acid sequences of TiI and castorA918:0-ACP desaturase cDNA (CAS) (ref. 11). Identical amino acids are indicated by colons. Amino acids which are absent relative to either of the two sequences are indicated by dashed lines. Alignment of the THI nucleotide sequence is maintained with a dotted line. The underlined alanine (amino acid 37) indicates the likely amino terminus of the mature peptide encoded by the type II cDNA. The high degree of amino acid sequence identity with the acid in transgenic tobacco callus does suggest possible bio- A&918:0-ACP desaturase, the apparent plastid localization of synthetic origins of the A6 double bond of petroselinic acid. the 36-kDa protein, and results of previous metabolic studies If the location of the double bond is determined by the (6) strongly suggest that the 36-kDa desaturase uses an position of carbon atoms from the methyl (or o) end of acyl acyl-ACP moiety as its in vivo substrate. Previously, oleic chains, then it is conceivable that the 36-kDa peptide is an 1 acid was the only plant fatty acid known to be synthesized via desaturase. As such, this enzyme could catalyze the w12 an acyl-ACP desaturase (7-9). desaturation of both palmitoyl (16:0)- and stearoyl (18:0)- An unexpected finding was the appearance of nearly equal ACP, resulting in A4-hexadecenoyl- and petroselinoyl-ACP, levels of A4-hexadecenoic acid and petroselinic acid in to- respectively. Alternatively, if double bond placement is bacco callus expressing the 36-kDa desaturase. Several hexa- decenoic acid isomers are detectable in extracts of coriander seed. One of these isomers has been identified by A B I, mass spectrometry as A4-hexadecenoic acid (unpublished data). However, amounts of this fatty acid are typically -<1% by weight of the total fatty acid of coriander seed extracts. In contrast, petroselinic acid accounts for .:70% by weight of 10 the total fatty acid of coriander seed (1). Despite our demonstration that the synthesis of petrose- linic acid involves a 36-kDa putative acyl-ACP desaturase, the immediate precursor of this fatty acid cannot be defini- tively identified from these experiments. However, the de- tection of A4-hexadecenoic acid in addition to petroselinic 2 5 4 16 A B C D 17 9 30 45 60 75 30 45 60 75 39 kWa Retention Time (min) Retention Time (min) - 36 kDa FIG. 4. Gas chromatograms of fatty acid methyl esters prepared from tobacco callus transformed with the pBI121 vector (A) and pB1l2l containing the coriander type II cDNA insert (B). Arrows FIG. 3. Western blot analysis of protein extracts from callus of indicate fatty acids present only in callus transformed with the type transgenic tobacco. Proteins of crude extracts were separated by II insert. The double-bond positions of A4-hexadecenoic acid SDS/11% PAGE, transferred to nitrocellulose, and probed with (16: 1A4) (peak 2) and petroselinic acid (18:1A6) (peak 5) were deter- polyclonal antibodies against the A918:0-ACP desaturase ofavocado. mined by GC-MS (see Fig. 5). Inset in B is an enlargement of peaks Lanes: A, coriander seed extract; B and C, extracts from two (peaks 5, 6, and 7) corresponding to the closely eluted isomers of independent samples of tobacco calli transformed with pBI121 18:1. Peak identification: 1, 16:0; 2, 16:1M&; 3, 17:0 (internal stan- containing the coriander type II cDNA; D, extract from tobacco dard); 4, 18:0; 5, 18:1A6; 6, 18:1A9; 7, 18:1A11; 8, 18:2; 9, 20:0; 10, callus transformed with the pBIl2l expression vector without insert. 18:3. Downloaded by guest on September 27, 2021 11188 Plant Biology: Cahoon et al. Proc. Nad. Acad. Sci. USA 89 (1992)

We thank Chris Somerville for support provided to this project and A 16:1 A4 CH3 critical reading of the manuscript. We thank Di Do for technical CHC(CH2)10CH CH(CH2)2C02CH3 assistance; Doug Gage and Zhi-Heng Huang for advice on prepara- 100 tion of fatty acid derivatives for GC-MS; Katherine Kun- 1Y7 2 15 Schmid, 1 15 1. CH3 x y Me imitsu Nakahira, and David Shintani for helpful suggestions; and Phil 362 Jensen for assistance with GC-MS. This work was supported in part -I by the Department of Agriculture/National Science Foundation/ loiI - -*-a X L . s .. L of 0 100 150 200 250 300 350 Department Energy Plant Science Center Program (Grant 88- 37271-3964). Acknowledgement is made to the Michigan Agricultural O0 Experiment Station for its support of this research. c 'aCO B 18:1Ln6 CH3 CH3(CH2),0CH ICH(H2)40C02CH3 1. Kleiman, R. & Spencer, G. F. (1982) J. Am. Oil Chem. Soc. 59, 100' 29-38. /7 Y 143 175 215 OH3 M 2. Ellenbracht, F., Barz, W. & Mangold, H. K. (1980) Planta 150, Y-32 y 390 114-119. 3. Dutta, P. C. & Appelqvist, L. A. (1991) Plant Sci. 75, 177-183. LA . b .,. .-A L 4. Browse, J. & Somerville, C. (1991) Annu. Rev. Plant Physiol. 100 200 300 Plant Mol. Biol. 42, 467-506. Mass/charge 5. Jaworski, J. G. (1987) in The Biochemistry of Plants, eds. Stumpf, P. K. & Conn, E. E. (Academic, New York), Vol. 9, FIG. 5. Mass of pp. 159-173. spectra thiomethyl adducts of fatty acid methyl 6. Cahoon, E. B. & Ohlrogge, J. B. (1991) INFORM 2, 342 esters detected exclusively in tobacco callus transformed with the (abstr.). type II coriander cDNA insert in pBI121. Shown are mass spectra of A4hexadecenoic acid (16:1A4) (peak 2, Fig. 4B) (A) and petroselinic 7. McKeon, T. A. & Stumpf, P. K. (1982) J. Biol. Chem. 257, acid 12141-12147. (18:1A6) (peak 5, Fig. 4B) (B) derivatives prepared from fatty 8. Jaworski, J. G. & Stumpf, P. K. (1974) Arch. Biochem. Bio- acid methyl esters of transgenic tobacco callus as described in the phys. 162, 158-165. text. 9. Nagai, J. & Bloch, K. (1968) J. Biol. Chem. 233, 4626-4633. 10. McKeon, T. A. & Stumpf, P. K. (1981) Methods Enzymol. 71, dictated by the position of carbon atoms from the carboxyl 275-281. (or A) end of acyl chains, then it is unlikely that the 36-kDa 11. Shanklin, J. & Somerville, C. (1991) Proc. Natd. Acad. Sci. peptide catalyzes both the A4 and A6 desaturation of palmi- USA 88, 2510-2514. toyl- and stearoyl-ACP, respectively. Instead, petroselinic 12. Laemmli, U. K. (1970) Nature (London) 227, 680-685. acid might result from the desaturation of a shorter-chain 13. Post-Beittenmiller, M. A., Schmid, K. M. & Ohlrogge, J. B. acyl-ACP by the 36-kDa peptide followed by elongation to (1989) Plant Cell 1, 889-899. petroselinoyl-ACP. For example, a possible biosynthetic 14. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual(Cold Spring Harbor Lab., Cold pathway might consist of the A4 desaturation of palmitoyl- Spring Harbor, NY). ACP to A4-hexadecenoyl-ACP with subsequent two-carbon 15. Short, J. M., Fernandez, J. M., Sorge, J. A. & Huse, W. D. elongation to petroselinoyl-ACP. (1988) Nucleic Acids Res. 16, 7583-7600. The synthesis of petroselinic acid and A4-hexadecenoic 16. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, acid in transgenic tobacco demonstrates the ability to pro- J. W. (1990) Methods Enzymol. 185, 60-89. duce new unsaturated fatty acids via gene-transfer technol- 17. Jefferson, R. A., Kavanaugh, T. A. & Bevan, M. W. (1987) ogy. This finding suggests the potential for the development EMBO J. 6,3901-3907. 18. Mersereau, M., Pazour, G. J. & Das, A. (1990) Gene 90, of a new plant oil (i.e., a high petroselinate oil) in an existing 149-151. oilseed crop. However, levels of petroselinic acid in the 19. Rogers, S. G., Horsch, R. B. & Fraley, R. T. (1986) Methods transgenic tobacco callus were 1% to 4% (wt/wt) of the total Enzymol. 118, 627-648. fatty acid. This rather low amount of petroselinic acid may 20. Morris, L. J., Wharry, D. M. & Hammond, E. W. (1967) J. suggest that other factors are required for high levels of Chromatogr. 31, 69-76. accumulation. 21. Yamamoto, K., Shibahara, A., Nakayama, T. & Kajimoto, G. Finally, the A918:O-ACP desaturase (11, 22) and the 36-kDa (1991) Chem. Phys. 60, 39-50. 22. G. desaturase of coriander share a high degree of amino acid Thompson, A., Scherer, D. E., Foxal-Van Aken, S., identity. The primary difference between the amino acid Kenny, J. W., Young, H. L., Shintani, D. K., Kirdl, J. C. & Knauf, V. C. (1991) Proc. Nat!. Acad. Sci. USA U, 2578-2582. sequences ofthese enzymes occurs near their amino termini. 23. Kinney, A. J., Hitz, W. D. & Yadav, N. S. (1990) in Plant The divergence in this region includes the absence of 15 LipidBiochemistry, Structure and Utilization, eds. Quinn, P. J. amino acids from the 36-kDa desaturase relative to the & Harwood, J. L. (Portland, London), pp. 126-128. A918:0-ACP desaturase. It is intriguing to speculate that the 24. Cheesbrough, T. M. & Cho, S. H. (1990) in Plant Lipid Bio- alteration in double-bond placement relates to this difference chemistry, Structure and Utilization, eds. Quinn, P. J. & Har- in the primary structures of these enzymes. wood, J. L. (Portland, London), pp. 129-130. Regardless, 25. Lutke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, comparative biochemical studies of the A918:0-ACP desatu- F. & Scheele, G. A. (1987) EMBO J. 6,43-48. rase and the 36-kDa desaturase of coriander may allow a 26. Keegstra, K., Olsen, L. J. & Theg, S. M. (1989) Annu. Rev. better understanding ofthe relationship between the catalytic Plant Physiol. Plant Mol. Biol. 40, 471-501. mechanism and the active sites of fatty acid desaturases. 27. Francis, G. W. (1981) Chem. Phys. Lipids 29, 369-374. Downloaded by guest on September 27, 2021