Expression of meadowfoam Des5 and FAE1 genes in yeast and in transgenic soybean somatic embryos,and their roles in fatty acid modification

Meadowfoam (Limnanthes spp.) species are unique in that their seeds are rich in the unusual fatty acids Δ⁵-eicosenoic acid (C20:1^Δ5) and the diene, C22:2^{Δ5, Δ13}. Previously the cloning of Δ⁵ desaturase (Des5) and fatty acid elongase 1 (FAE1) meadowfoam genes and their expression in soybean were reported. Here, we present the first successful expression of the Limnanthes Des5 in yeast, resulting in the desaturation of C16:0, C18:0 and C20:0 to their corresponding cis Δ⁵ isomers. In soybean (Glycine max L.), Limnanthes Des5/FAE1 double transformant somatic embryos fed with radiolabeled C14:0 or C16:0 could elongate these substrates to C18:0, C20:0 and C22:0 and C24:0. However, radiolabeled C18:1^Δ9 or C20:1^Δ11 were not elongated to their respective monounsaturated very long-chain products, confirming that the cloned Limnanthes FAE1 homolog gene product was specific for elongating saturated fatty acids. To understand better the biosynthetic pathway for C22:2^{Δ5, Δ13}, soybean somatic embryos transformed with the Des5 cDNA were fed in culture with 〚1-¹⁴C〛C 22:1^Δ13 fatty acid, which resulted in the biosynthesis of 〚1-¹⁴C〛-labeled C22:2^{Δ5, Δ13}. Cell-free preparations enriched with detergent-solubilized Δ⁵ desaturase activity extracted from both developing meadowfoam seeds and from Des5 transgenic soybean embryos, produced ¹⁴C-22:2^{Δ5, Δ13} when supplied with 〚1-¹⁴C〛 C22:1-CoA. Thus, both the in vivo and in vitro experiments showed that the biosynthesis of C22:2^{Δ5, Δ13} can occur in somatic soybean embryos transformed with the Limnanthes Des5 cDNA, and confirmed that the pathway for C22:2 biosynthesis in meadowfoam involves further desaturation of erucoyl-CoA by a Δ⁵-regiospecific desaturase.     

Production of 22:2<Superscript>Δ5,Δ13</Superscript> and 20:1<Superscript>Δ5</Superscript> in <Emphasis Type="Italic">Brassica carinata</Emphasis> and soybean breeding lines via introduction of <Emphasis Type="Italic">Limnanthes</Emphasis> genes

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1⁵ and the diene 22:2^5,13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2^5,13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1⁵ to approximately 10% of the total fatty acids of seeds. The content of C₂₀ and C₂₂ fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2^5,11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C₂₀ and C₂₂ fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1³, respectively.     

Dimorphecolic acid is synthesized by the coordinate activities of two divergent Delta12-oleic acid desaturases

Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species. This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation. Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid. cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds. Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer. When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid. When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid [corrected]. These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid. Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2. The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.     

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.     

Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase

The marine parasitic protozoon Perkinus marinus synthesizes the polyunsaturated fatty acid arachidonic acid via the unusual alternative Delta8 pathway in which elongation of C18 fatty acids generates substrate for two sequential desaturations. Here we have shown that genes encoding the three P. marinus activities responsible for arachidonic acid biosynthesis (C18 Delta9-elongating activity, C20 Delta8 desaturase, C20 Delta5 desaturase) are genomically clustered and co-transcribed as an operon. The acyl elongation reaction, which underpins this pathway, is catalyzed by a FAE1 (fatty acid elongation 1)-like 3-ketoacyl-CoA synthase class of condensing enzyme previously only reported in higher plants and algae. This is the first example of an elongating activity involved in the biosynthesis of a polyunsaturated fatty acid that is not a member of the ELO/SUR4 family. The P. marinus FAE1-like elongating activity is sensitive to the herbicide flufenacet, similar to some higher plant 3-ketoacyl-CoA synthases, but unable to rescue the yeast elo2Delta/elo3Delta mutant consistent with a role in the elongation of polyunsaturated fatty acids. P. marinus represents a key organism in the taxonomic separation of the single-celled eukaryotes collectively known as the alveolates, and our data imply a lineage in which ancestral acquisition of plant-like genes, such as FAE1-like 3-ketoacyl-CoA synthases, occurred via endosymbiosis. The P. marinus FAE1-like elongating activity is also indicative of the independent evolution of the alternative Delta8 pathway, distinct from ELO/SUR4-dependent examples.     

Fungal responsive fatty acid acetylenases occur widely in evolutionarily distant plant families

The fungal elicitor-induced ELI12 gene from parsley has been previously shown to encode a divergent form of the Delta12-oleic acid desaturase. In this report, we show that the ELI12 gene product is a fatty acid acetylenase or a triple-bond-forming enzyme. Expression of this enzyme in transgenic soybean seeds was accompanied by the accumulation of the Delta12-acetylenic fatty acids, crepenynic and dehydrocrepenynic acids. Using PCR with degenerate oligonucleotides, we also show that homologs of the ELI12 gene are present in other members of the Apiaceae family. In addition, cDNAs for divergent forms of the Delta12-oleic acid desaturase were detected among the expressed sequence tags (ESTs) from English ivy, an Araliaceae species, and sunflower, an Asteraceae species. As with the ELI12 gene, expression of these cDNAs in transgenic soybean embryos was accompanied by the accumulation of crepenynic and dehydrocrepenynic acids. Homologs of the sunflower acetylenase gene were also detected in other Asteraceae species, as revealed by PCR analysis of isolated genomic DNA. Results from Northern blot and EST analyses indicated that the expression of the sunflower gene, like ELI12, was induced by fungal elicitation. Overall, these results demonstrate that expressed genes for Delta12-fatty acid acetylenases occur in at least three plant families, and are responsive to fungal pathogenesis. Natural products derived from crepenynic and dehydrocrepenynic acids that display antifungal, insecticidal, and nematicidal properties are distributed through at least 15 plant families. The acetylenases described here provide probes for chemotaxonomists, and facilitate functional genomic and molecular investigations of these defensive mechanisms.     

Acyl-CoA Z9- and Z10-desaturase genes from a New Zealand leafroller moth species,Planotortrix octo

Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR. The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9). The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids. Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T. ni and Helicoverpa zea (57 and 56%, respectively). The ORFs of these two P. octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae. Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids. Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid. The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase.     

Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation

Omega6- and omega3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Delta6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Delta6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Delta6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.     

New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.     

Expression of the Arabidopsis ADS1 gene in Brassica juncea results in a decreased level of total saturated fatty acids

Brassica juncea plants transformed with the Arabidopsis ADS1 gene, which encodes a plant homologue of the mammalian and yeast acyl-CoA Delta9 desaturases and the cyanobateria acyl-lipid Delta9 desaturase, were found to have a statistically significant decrease in the level of saturated fatty acids in seeds. The decrease in the level of saturated fatty acids is largely attributable to decreases in palmitic acid (16:0) and stearic acid (18:0), although arachidic acid (20:0), behenic acid (22:0) and lignoceric acid (24:0) were also decreased in the transgenic seeds compared to the negative control lines. As a result, the level of oleic acid (18:1) was slightly increased in the transgenic seed lines compared to the non-transformed controls. However, a decrease in saturated fatty acid is not always accompanied by the corresponding increase in mono-unsaturated fatty acids. For example, palmitoleic acid (16:1), gondoic acid (20:1) and nervonic acid (24:1) were all found to be decreased in transgenic seeds. The levels of linoleic acid (18:2) and linolenic acid (18:3) were also notably changed in the transgenic lines compared to the controls. The present study provides preliminary experimental data suggesting that the Arabidopsis ADS1 encodes a fatty acid Delta9 desaturase and could be useful in genetic engineering for modifying the level of saturated fatty acids in oilseed crops. However, the effect of ADS1 gene expression on seed oil fatty acid composition is beyond the changes of total saturated and mono-unsaturated fatty acids, which suggests a complex mechanism is involved in the regulation of fatty acid metabolism.     

Effects of fenofibrate and insulin on the biosynthesis of unsaturated fatty acids in streptozotocin diabetic rats

Both insulin and PPAR-alpha up-modulate hepatic Delta9, Delta6 and Delta5 desaturating enzymes involved in the biosynthesis of mono- and polyunsaturated fatty acids. Currently, we have examined for 9 days the independent and simultaneous effects of daily glargine insulin and fenofibrate administration on the insulinemia, glycemia, hepatic acyl-CoA oxidase activity and mRNAs and enzymatic activities of stearoyl-CoA desaturase-1 (SCD-1) and Delta5 desaturase in streptozotocin diabetic rats. Glargine insulin depressed the hyperglycemia of diabetic rats at 4h, but not after 24h of injection. Fenofibrate increased the radioimmunoreactive insulinemia in non-diabetic rats without changing the glycemia. Insulin increased the mRNAs and activities of SCD-1 and Delta5 desaturase depressed in diabetic rats. Fenofibrate increased acyl-CoA oxidase activity, and the mRNAs and activities of both desaturating enzymes in non-diabetic, diabetic and insulin-treated diabetic rats, but was less effective in the mRNAs modification of diabetic animals. Therefore, insulin, and fenofibrate through PPAR-alpha activation, enhance liver mRNAs and activities of SCD-1 and Delta5 desaturases independently and synergistically through different mechanisms. Insulin and fenofibrate independently increased the 18:1/18:0 ratio in liver lipids, increasing the fluidity of the membranes. The 20:4/18:2 ratio was maintained. Fenofibrate increased palmitic acid, but decreased stearic acid percentage in liver lipids.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids

Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.     

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.