Sex pheromone biosynthesis in the processionary moth Thaumetopoea pityocampa by delta-13 desaturation

In vivo treatments of female sex pheromone glands of the processionary moth, Thaumetopoea pityocampa, with mass-labeled fatty acids showed that (Z)-13-hexadecen-11-ynyl acetate, the main sex pheromone component, is biosynthesized from palmitic acid by the combined action of delta-11 and delta-13 desaturases. The involvement of this unusual delta-13 has been proven by application of [16,16,16-2H3] [1,2-13C2]-hexadecanoic acid to the glands with a resultant incorporation of all labeled atoms into the pheromone and each one of the corresponding intermediates. These results seem to exclude alternative biosynthetic pathways, such as chain shortening and elongation combined with delta-11 desaturation. The delta-11 desaturase responsible for the formation of the triple bond in both the 11-hexadecynoyl and (Z)-13-hexadecen-11-ynoyl intermediates is also an unusual enzyme not previously reported in lepidopteran sex pheromone biosynthesis.     

Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.     

Thiafatty acids as tracers to investigate biosynthetic pathways of lepidopteran sex pheromones

In order to investigate the potential utility of thiafatty acids as tracers for biosynthetic studies of moth sex pheromones, a series of thiatetradecanoic acids, namely 8-, 9-, 10-, 11-, 12- and 13-thiatetradecanoic, were prepared and their metabolism was investigated in pheromone glands of Spodoptera littoralis. Analysis by gas chromatography coupled to mass spectrometry of extracts from pheromone glands treated with the above acids showed that only 8-thiatetradecanoic acid and 13-thiatetradecanoic acid were metabolized by desaturation and were incorporated into the sex pheromone biosynthetic pathway. 13-Thiatetradecanoic acid was converted into (E)- and (Z)-13-thiatetradec-11-enoic acids, (Z,E)-13-thiatetradeca-9,11-dienoic acid, 11-thiadodecanoic acid, (E)- and (Z)-11-thiadodec-9-enoic acids and 15-thiahexadecanoic acid. 8-Thiatetradecanoic acid gave rise to two monoenoic thiafatty acids and two dienoic thiafatty acids, which were assigned to (Z)- and (E)-8-thiatetradec-11-enoic acids, (Z,E)-8-thiatetradeca-9,11-dienoic acid and (E,E)-8-thiatetradeca-10,12-dienoic acid. The other thiafatty acids tested, 9-, 10-, 11- and 12-thiatetradecanoic acids, were not metabolized by desaturation, although the corresponding products of beta-oxidation and chain elongation were detected. The occurrence of sulfoxides was not detected in this case, in disagreement with results on the metabolism of some thiaacids previously reported by other authors in yeast, Saccharomyces cerevisiae.     

Neofunctionalization in an ancestral insect desaturase lineage led to rare Δ6 pheromone signals in the Chinese tussah silkworm

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ⁶ and Δ¹¹ regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ⁶ and Δ¹¹ desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ⁶ desaturase involved in insect mate communication. The occurrence of this novel Δ⁶ desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ¹¹ desaturases commonly involved in moth pheromone biosynthesis.     

Fatty acid and sex pheromone changes and the role of glandular lipids in the Z-strain of the European corn borer, Ostrinia nubilalis (Hübner)

Lipids in the sex pheromone gland of females of the Z-strain of Ostrinia nubilalis were analyzed for fatty acyl pheromone analogs (FAPAs) and other potential biosynthetic intermediates. More than 80% of the FAPAs were found in the triacylglycerols (TGs), with smaller amounts found in the phosphatidyl cholines, ethanolamines, and serines. Analysis of the TGs by lipase revealed that the two FAPAs were distributed fairly evenly among all three stereospecific positions. Comparison of changes in titers of key glandular fatty acids with those of pheromone components, with respect to photoperiodic time and age of females, showed that both FAPA and pheromone titers exhibited a cyclical pattern with peaks in the scotophase and valleys in the photophase. However, whereas pheromone titer tended to peak in the first half of the scotophase, FAPA titer peaked at the end of the scotophase. Significantly, the titer of the FAPA of the minor component, (E)-11-tetradecenyl acetate (3% of pheromone), was always much greater than the titer of the FAPA of the major component, (Z)-11-tetradecenyl acetate (97%), of the pheromone. Titer of myristate, an intermediate in pheromone biosynthesis, was also higher during the scotophase than the photophase. However, myristate titer showed a pronounced dip in the middle of the scotophase. These data suggest two roles for glandular lipids in sex pheromone biosynthesis in O. nubilalis. Firstly, they remove excess FAPA of the minor component so the fatty acid reductase system is not presented with a high ratio of this isomer (which would otherwise result from the reductase's own selectivity), which could cause changes in the final pheromone ratio. Secondly, hydrolysis of the large amounts of stored saturated fatty acids from the TGs may provide substrate for pheromone biosynthesis.     

Inhibition of the acyl-CoA desaturases involved in the biosynthesis of Spodoptera littoralis sex pheromone by analogs of 10,11-methylene-10-tetradecenoic acid

The desaturase inhibitory activity of the cyclopropenyl alcohols 9,10-methylene-9-tetradecen-1-ol (9-MTOL), 10,11-methylene-10-tetradecen-1-ol (10-MTOL) and 11,12-methylene-11-tetradecen-1-ol (11-MTOL), which are structural analogs of 10,11-methylene-10-tetradecenoic acid (10-MTA), is reported. At equimolar ratios with respect to the different substrates, the three compounds completely inhibited the three desaturation reactions involved in the biosynthesis of Spodoptera littoralis sex pheromone. The dose-dependence of inhibition was determined for 10-MTA and its alcohol derivative. Both compounds inhibited the transformation of perdeuterated palmitic acid into perdeuterated (Z)-11-hexadecenoic acid and that of (E)-11-tridecenoic acid into (Z,E)-9,11-tridecadienoic acid with similar IC(50) values. The overall results presented in this work support scattered data that neither the free carboxyl groups nor their acyl-CoA esters are a requisite for inhibition of desaturases. Since the synthesis of cyclopropenols is much more convenient than that of cyclopropene fatty acids, this finding is of economical relevance regarding the putative use of cyclopropene derivatives in pest control.     

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis

In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Deltaelo1/Deltaole mutants, which are both elongase 1 and Delta9 desaturase-deficient, were transformed with the S. littoralis Delta11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Delta11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Delta11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Delta11 desaturase of S. littoralis is a bifunctional enzyme with both Delta11 and Delta10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.     

Stereospecificity of the (Z)-9 desaturase that converts (E)-11-tetradecenoic acid into (Z,E)-9,11-tetradecadienoic acid in the biosynthesis of Spodoptera littoralis sex pheromone

Moth pheromone glands contain desaturases that catalyze the formation of conjugated dienoic fatty acids. In this article we present the first stereochemical study on one of these enzymes, namely the Delta(9) desaturase of (E)-11-tetradecenoic acid, using the moth Spodoptera littoralis as a biological model and enantiopure deuterated probes derived from tridecanoic acid. Gas chromatography coupled to mass spectrometry analysis of methanolyzed lipidic extracts from glands incubated with each individual probe showed that in the transformation of (E)-11-tetradecenoic acid into (Z,E)-9,11-tetradecadienoic acid both pro-(R) hydrogen atoms at C9 and C10 are removed from the substrate.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

Sex pheromone biosynthesis in the tortricid moth Planotortrix excessana (Walker) involves chain-shortening of palmitoleate and oleate

Biosynthesis of the sex pheromone components, (Z)-5-tetradecenyl acetate (Z5-14:OAc) and (Z)-7-tetradecenyl acetate (Z7-14:OAc), was investigated in the New Zealand tortricid moth Planotortrix excessana (Walker) by fatty acid methyl ester (FAME) analysis of base-methanolyzed extracts of lipids in the sex pheromone gland and through application of various labelled fatty acids. Analysis of the base-methanolyzed gland extracts revealed common FAMEs, including methyl oleate and methyl palmitoleate, as well as the FAMEs of the putative precursors, methyl (Z)-5-tetradecenoate and methyl (Z)-7-tetradecenoate. Application of labelled, saturated fatty acids, myristic, palmitic, and stearic did not result in any significant incorporation of label into either of the unsaturated pheromone components, although label was incorporated into tetradecyl acetate (14:OAc). In contrast, application of labelled oleic acid resulted in incorporation of label into Z5-14:OAc but not into Z7-14:OAc or into 14:OAc, whereas application of labelled palmitoleic acid resulted in incorporation of label into Z7-14:OAc but not into Z5-14:OAc or 14:OAc. These data support a route for biosynthesis of Z5-14:OAc and Z7-14:OAc in this species by limited β-oxidation of the common fatty acyl moieties, respectively, oleate (involving two cycles of 2-carbon chain-shortening) and palmitoleate (involving only one cycle of 2-carbon chain-shortening), and apparently involving no desaturase (other than the common Δ9) specific to sex pheromone biosynthesis. Interestingly, P. excessana females biosynthesize the same component (Z5-14:OAc) from an entirely different route from that of the related species Ctenopseustis obliquana (which biosynthesizes Z5-14:OAc by Δ5-desaturation of myristate). Additionally, the pheromone biosynthesis activating neuropeptide (PBAN) stimulates pheromone biosynthesis in this species. Arch. Insect Biochem. Physiol. 37:158–167, 1998. © 1998 Wiley-Liss, Inc.     

Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis virescens

Sex pheromones of many moth species have relatively simple structures consisting of a hydrocarbon chain with a functional group and one to several double bonds. These sex pheromones are derived from fatty acids through specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled tetradecanoic, hexadecanoic, and octadecanoic acid precursors into pheromone components of Heliothis subflexa and Heliothis virescens. The two species utilize (Z)11-hexadecenal as the major pheromone component, which is produced by Delta11 desaturation of hexadecanoic acid. H. subflexa also produced (Z)11-hexadecanol and (Z)-11-hexadecenyl acetate via Delta11 desaturation. In H. subflexa, octadecanoic acid was used to biosynthesize the minor pheromone components (Z)9-hexadecenal, (Z)9-hexadecenol, and (Z)9-hexadecenyl acetate. These minor components are produced by Delta11 desaturation of octadecanoic acid followed by one round of chain-shortening. In contrast, H. virescens used hexadecanoic acid as a substrate to form (Z)11-hexadecenal and (Z)11-hexadecenol and hexadecenal. H. virescens also produced (Z)9-tetradecenal by Delta11 desaturation of the hexadecanoic acid followed by one round of chain-shortening and reduction. Tetradecanoic acid was not utilized as a precursor to form Z9-14:Ald in H. virescens. This labeling pattern indicates that the Delta11 desaturase is the only active desaturase present in the pheromone gland cells of both species.     

Comparative study of sex pheromone composition and biosynthesis in Helicoverpa armigera, H. assulta and their hybrid

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.     

Identification of a four-component sex pheromone blend in Bonagota cranaodes (Lepidoptera: Tortricidae)

Analysis of sex pheromone glands of the apple leafroller Bonagota cranaodes Meyrick by gas chromatography coupled with mass spectrometry or electroantennographic detection showed the presence of 14 structurally related acetates and alcohols of the chain length 10-18, including the main pheromone component (E,Z)-3,5-dodecadienyl acetate (E3,Z5-12Ac). Male antennae responded to the main compound, its Z,Z isomer, (E,Z) -3,5-tetradecadienyl acetate (E3,Z5-14Ac), and the monoenes (Z)-5-dodecenyl acetate (Z5-12Ac) and (Z)-9-hexadecenyl acetate (Z9-16Ac). Traps baited with a four-component blend of E3,Z5-12Ac, Z5-12Ac, E3,Z5-14Ac, and Z9-16Ac in a 100:5:5:100 ratio were significantly more attractive than the main compound alone. This improved trap lure is more suitable for monitoring population densities of B. cranaodes, and for detection of the onset of the seasonal flight period. A more complete pheromone blend is of importance also with respect to current attempts to develop mating disruption for control of this major pest of apple in Brazil.     

Mimicking Insect Communication: Release and Detection of Pheromone,Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered.     

Wind tunnel studies on the effects of secondary sex pheromone components on the behaviour of male Egyptian cotton leafworm moths, Spodoptera littoralis

ABSTRACT. The responses of adult male Spodoptera littoralis (Boisd.) (Noctuidae) to components of the female sex pheromone were examined in a wind tunnel. The responses of male moths from both Egyptian and Cretan stocks to the primary 'attractant' (Z, E)-9,11 tetradecadienyl acetate (III), were compared with the responses to III combined with various percentages of the secondary components tetradecyl acetate (I), (Z)-9-tetradecenyl acetate (IIA), (E)-11-tetradecenyl acetate (IIB), IIA and IIB combined, and(Z, E)-9,12-tetradecadienyl acetate (IV). Analysis of the flight behaviour recorded on videotape showed that with one exception (IV), the secondary components increased the males' success in reaching the source and increased the length of time they spent at the source. In most cases, increasing the relative percentage of the secondary component caused a reduction in the number of males responding. The results are compared with those obtained in field trials with S.littoralis.     

CONTROL OF SEX PHEROMONE BIOSYNTHETIC PATHWAY BY PBAN IN ASIAN CORN BORER OSTRINIA FURNACALlS*

Abstract Sex pheromone titer in Ostrinia furnacalis was significantly decreased to a very low level by decapitation, but it could be restored by injection of head extract prepared from both male and female moths or synthetic pheromone biosynthesis activating neuropepide (PBAN). This fact indicates that pheromone production is under the control of a PBAN-like factor. The sex pheromone biosynthetic pathway of O. furnacalis originates with the biosynthesis of palmitic acid and followed by A14 desaturation, chain shortening, reduction and acetylation to form the pheromone components, (Z) and (E)-12-tetradecenyl acetate. In order to determine which step in the pathway is controlled by PBAN, the incorporation of different labeled precursors into the pheromone and its intermediate were studied. Our results suggest that PBAN controls pheromone biosynthesis in O. furnacalis by mainly regulating an early step from acetate to palmitic acid.     

Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide

(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by Delta(11)-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluorobenzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass spectrometry is also reported.     

Comparative sex pherome biosynthesis in Thaumetopoea pityocampa and T. processionea: a rationale for the phenotypic variation in the sex pherome within the genus Thaumetopoea

The female sex pheromones of the Mediterranean processionary moths (Thaumetopoea sp.) are conjugated dienes or enynes of 16 carbon atoms with the unsaturations located at C11 and C13. To investigate the biochemical basis of this phenotypic variation, the biosynthetic pathway of T. processionea sex pheromone, a diene acetate, has been elucidated and compared to that reported for the enyne-producing species T. pityocampa. Mass labeling experiments showed that T. processionea sex pheromone is biosynthesized from palmitic acid, by subsequent (Z)-11 and (Z)-13 desaturations and final reduction and acetylation. The Pheromone Biosynthesis Activating Neuropeptide (PBAN) activates this biosynthetic pathway downstream of the dienoate intermediate. When either 11-hexadecynoic acid or (Z)-13-hexadecen-11-ynoic acid were administered to T. processionea, this species was able to produce the enyne sex pheromone of T. pityocampa upon PBAN stimulation. In contrast, T. pityocampa does not produce either 11-hexadecynyl acetate or (Z,Z)-11,13-hexadecadienyl acetate, despite having the corresponding precursors in the pheromone gland. However, both acetates are detected after administration of the corresponding alcohols. These overall results suggest that the absence of delta(11) acetylenase and the existence of an enynoate specific reductase in the diene and enyne-producing Thaumetopeae, respectively, account for the different sex pheromones produced by the two groups.     

Metabolic Evidence for the Involvement of a [delta]4-Palmitoyl-Acyl Carrier Protein Desaturase in Petroselinic Acid Synthesis in Coriander Endosperm and Transgenic Tobacco Cells

We have previously demonstrated that the double bond of petroselinic acid (18:1[delta]6cis) in coriander (Coriandrum sativum L.) seed results from the activity of a 36-kD desaturase that is structurally related to the [delta]9-stearoyl-acyl carrier protein (ACP) desaturase (E.B. Cahoon, J. Shanklin, J.B. Ohlrogge [1992] Proc Natl Acad Sci USA 89: 11184-11188). To further characterize the biosynthetic pathway of this unusual fatty acid, 14C-labeling experiments were conducted using developing endosperm of coriander. Studies were also performed using suspension cultures of transgenic tobacco (Nicotiana tabacum L.) that express the coriander 36-kD desaturase, and as a result produce petroselinic acid and [delta]4-hexadecenoic acid. When supplied exogenously to coriander endosperm slices, [1-14C]palmitic acid and stearic acid were incorporated into glycerolipids but were not converted to petroselinic acid. This suggested that petroselinic acid is not formed by the desaturation of a fatty acid bound to a glycerolipid or by reactions involving acyl-coenzyme As (CoA). Instead, evidence was most consistent with an acyl-ACP route of petroselinic acid synthesis. For example, the exogenous feeding of [1-14C]lauric acid and myristic acid to coriander endosperm slices resulted in the incorporation of the radiolabels into long-chain fatty acids, including primarily petroselinic acid, presumably through acyl-ACP-associated reactions. In addition, using an in vitro fatty acid biosynthetic system, homogenates of coriander endosperm incorporated [2-14C]malonyl-CoA into petroselinic acid, of which a portion was detected in a putative acyl-ACP fraction. Furthermore, analysis of transgenic tobacco suspension cultures expressing the coriander 36-kD desaturase revealed significant amounts of petroselinic acid and [delta]4-hexadecenoic acid in the acyl-ACP pool of these cells. Also presented is evidence derived from [U-14C]nonanoic acid labeling of coriander endosperm, which demonstrates that the coriander 36-kD desaturase positions double bonds relative to the carboxyl end of acyl-ACP substrates. The data obtained in these studies are rationalized in terms of a biosynthetic pathway of petroselinic acid involving the [delta]4 desaturation of palmitoyl-ACP by the 36-kD desaturase followed by two-carbon elongation of the resulting [delta]4-hexadecenoyl-ACP.