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Keywords:

  • acetylenase;
  • desaturase;
  • fungal elicitation;
  • secondary metabolism;
  • crepenynic acid

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The fungal elicitor-induced ELI12 gene from parsley has been previously shown to encode a divergent form of the Δ12-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 Δ12-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 Δ12-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 Δ12-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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Δ12-oleic acid desaturase or FAD2 catalyzes the introduction of a double bond at the Δ12 position of oleic acid (18:1Δ9) to form linoleic acid (18:2Δ9,12). Thus, this enzyme plays a key role in the production of polyunsaturated fatty acids for membranes and seed oils of plants. While FAD2 is found throughout the plant kingdom, a number of functionally and structurally divergent forms of this enzyme have been identified in seeds of plants that accumulate a variety of unusual fatty acids. Divergent forms of FAD2 identified to date include fatty acid hydroxylases, epoxygenases, triple-bond-forming enzymes or acetylenases, and conjugated double-bond-forming enzymes or conjugases (Voelker and Kinney, 2001). These enzymes, unlike the ‘typical’ FAD2, occur only in restricted taxa.

A cDNA for a divergent form of FAD2 was previously identified in parsley (Petroselinum crispum), an Apiaceae species (Kirsch et al., 1997a). Expression of the corresponding gene, which was designated as ELI12, was found to be induced by fungal elicitation of the parsley suspension cultures (Somssich et al., 1989). Attempts to elucidate the function of the ELI12 gene product, however, were not successful. In these studies, expression of the typical FAD2 from parsley in Saccharomyces cerevisiae was accompanied by the accumulation of linoleic acid in the transformed cells (Kirsch et al., 1997a). By contrast, expression of the ELI12 cDNA had no detectable effect on the fatty acid composition of the recombinant yeast cells (Kirsch et al., 1997a). Like ELI12, expression of the typical FAD2 gene, as well as the Δ15-linoleic acid desaturase gene (or FAD3), is upregulated by fungal elicitation of parsley leaves and suspension cells (Kirsch et al., 1997a,b). The induction of FAD2 and FAD3 expression may be associated with the production of polyunsaturated fatty acids that are used in the maintenance of the membranes following fungal pathogenesis, or as substrates for the synthesis of signaling compounds such as jasmonic acid (Reymond and Farmer, 1988). However, given that the function of the divergent FAD2 encoded by ELI12 has not been previously determined, its role in the response of parsley cells to fungal infection cannot be speculated.

There has been considerable recent interest in divergent forms of FAD2, particularly with regard to their role in the biosynthesis of unusual fatty acids that accumulate to high levels in seed oils of certain species (Voelker and Kinney, 2001). Fatty acids that arise from reactions catalyzed by these divergent FAD2s include hydroxy (van de Loo et al., 1995), epoxy, and acetylenic or triple-bond-containing fatty acids (Lee et al., 1998). The identification of ELI12 in parsley, however, is puzzling. Parsley is not known to accumulate significant amounts of any unusual fatty acids that would be synthesized by a divergent form of FAD2. Furthermore, no close phylogenetic relationship is evident between the ELI12 gene product and any of the previously identified divergent FAD2 polypeptides (Kirsch et al., 1997a).

The apparently anomalous occurrence of ELI12 in parsley and its lack of close identity with the other FAD2-related enzymes sparked our curiosity. Studies were thus undertaken to establish the function of the ELI12 gene product. As described here, we have determined that this enzyme is a fatty acid acetylenase that generates acetylenic or triple-bond-containing fatty acids when expressed in transgenic soybean seeds. In addition, we show that the FAD2-related acetylenases also occur in the Araliaceae and Asteraceae families, including species that are not known to accumulate unusual fatty acids associated with the activity of a divergent FAD2. We also show that, like ELI12, the expression of the FAD2 acetylenase gene from sunflower (Helianthus annuus), an Asteraceae species, is induced by fungal elicitation. The fatty acid products of the acetylenases described in this study are precursors of biologically active (e.g. antifungal) polyacetylenic compounds that are known to occur in members of the Apiaceae, Araliaceae, and Asteraceae families (Barley et al., 1988; Bu'Lock and Smith, 1967). This points to a role of the FAD2 acetylenases in the biosynthetic pathways of the secondary defense compounds, including those that are upregulated in response to fungal elicitation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Functional identification of the ELI12 gene product

The ELI12 gene was originally shown to correspond to an RNA transcript that was upregulated in response to fungal elicitation of the parsley suspension cells (Somssich et al., 1989). This gene was later found to encode a Δ12-oleic acid desaturase (or FAD2)-related polypeptide with 61% amino acid sequence identity with the Arabidopsis FAD2 (Kirsch et al., 1997a). By contrast, the ‘typical’ FAD2 from parsley shares 74% identity with the Arabidopsis FAD2. Attempts to identify the function of the ELI12 polypeptide by expression in S. cerevisiae did not result in any detectable alteration in the fatty acid composition of the yeast cells (Kirsch et al., 1997a). Expression of the typical FAD2 from parsley, however, was accompanied by the conversion of oleic acid to linoleic acid in the yeast cells, which was consistent with the activity of a FAD2 enzyme (Kirsch et al., 1997a). The inability of the ELI12 gene product to alter the fatty acid composition of S. cerevisiae is similar to what has been observed for certain functional classes of divergent FAD2s. For example, expression of FAD2 acetylenases and epoxygenases in yeast typically results in little or no detectable fatty acid products (Lee et al., 1998). Furthermore, FAD2 and divergent forms of FAD2 are typically recalcitrant to direct assay of membrane extracts from plant and recombinant yeast (Shanklin and Cahoon, 1998).

As an alternative approach for identifying its activity, the ELI12 polypeptide was expressed in seeds of transgenic soybean. In these experiments, the open-reading frame of ELI12 was amplified by PCR from the genomic DNA of parsley and was linked to a strong seed-specific promoter. High levels of expression of ELI12 were detected in developing seeds of the transgenic plants (Figure 1). In addition, two novel fatty acids were identified in mature seeds from these plants. The two fatty acids, which are labeled as ‘Acet1’ and ‘Acet2’ in Figure 2(b), composed 3–4 wt% of the total fatty acids of the transgenic seeds (Table 1). Neither fatty acid was detected in seeds from the non-transformed plants. The methyl esters of Acet1 and Acet2 displayed gas chromatographic retention times that were identical to those of the methyl esters of the two acetylenic fatty acids (Figure 2). The methyl ester of Acet1, for example, displayed a retention time identical to that of the methyl ester of crepenynic acid (12-acetylenic-18:1Δ9cis) isolated from the seeds of Hawk's beard (Crepis rubra). In addition, the retention time of the Acet2 methyl ester was the same as that of the dehydrocrepenynic acid (12-acetylenic-18:2Δ9cis,14cis) methyl ester standards, which were isolated from chanterelle mushroom (Cantharellus cibarius) lipids and were prepared by chemical synthesis (Zhu and Minto, 2001). Furthermore, the mass spectra of the Acet1 and Acet2 methyl esters were indistinguishable from those of methyl crepenynate and dehydrocrepenynate, respectively (Figure 3). These results are thus consistent with the identification of Acet1 and Acet2 in the ELI12-expressing soybean seeds as crepenynic and dehydrocrepenynic acids, respectively.

image

Figure 1. Expression of the parsley ELI12 gene in developing soybean seeds transformed with this gene (+ELI12) or in developing seeds from non-transformed plants (Null).

(a) Total RNA (10 µg) from developing soybean seeds was electrophoresed, blotted, and then hybridized with a 32P-radiolabeled probe derived from the open-reading frame of ELI12.

(b) The ethidium bromide-stained gel corresponding to the Northern blot.

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image

Figure 2. Fatty acid composition of soybean seeds from non-transformed plants (Null) or plants transformed with ELI12 under control of a strong seed-specific promoter (+ELI12).

(a) Gas chromatograms of fatty acid methyl esters prepared from mature soybean seeds. Novel fatty acids in seeds of ELI12 transformants are indicated as Acet1 and Acet2. The other labeled peaks correspond to methyl esters of the following fatty acids: palmitic acid (16:0); stearic acid (18:0); oleic acid (18:1); linoleic acid (18:2); and linolenic acid (18:3).

Std1 and Std2 are gas chromatograms of standards consisting of the methyl esters of crepenynic acid (Std1) and 14-cis-dehydrocrepenynic acid (Std2).

(b) Chemical structures of crepenynic and dehydrocrepenynic acids.

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Table 1.  Fatty acid composition of non-transformed soybean seeds and transgenic soybean seeds expressing the parsley ELI12 gene
Fatty acidWeight percentage of total fatty acids
Untransformed (n = 7)+ELI12 (n = 10)
  • Values are presented as weight percentage of the total fatty acids of seeds, and were obtained from independent measurements of 7–10 single seeds (±SD) from each line.

  • 1

    N.D., Not detected.

  • 2

    Includes 20:0 and 22:0.

16:011.7 ± 0.911.8 ± 0.9
18:03.6 ± 0.23.5 ± 0.3
18:110.8 ± 1.513.5 ± 2.4
18:259.1 ± 1.657.3 ± 2.2
18:314.5 ± 2.010.0 ± 2.6
CrepenynicN.D.11.4 ± 0.4
DehydrocrepenynicN.D.2.2 ± 0.6
Other2<1.0<1.0
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Figure 3. Mass spectral identification of acetylenic fatty acid methyl esters from transgenic soybean seeds expressing ELI12.

Shown are mass spectra of fatty acid methyl esters corresponding to Acet1 (a) and Acet2 (b) from soybean seeds expressing ELI12. Also shown are mass spectra of methyl esters of crepenynic acid (c) and dehydrocrepenynic acid (d), which were isolated from extracts of Crepis rubra seeds and Cantharellus cibarius mushrooms, respectively. The mass spectra of Acet1 and Acet2 are consistent with those of methyl esters of crepenynic and dehydrocrepenynic acid, respectively.

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Nuclear magnetic resonance studies were conducted to provide further structural characterization of Acet2 (Figure 2) produced by transgenic soybean seeds expressing ELI12. The phenacyl ester derivative of Acet2 was prepared from transgenic soybean seeds and was purified by HPLC. The phenacyl derivative of Acet2 displayed the same retention time as the phenacyl derivative of dehydrocrepenynic acid purified from chanterelle mushrooms. Additionally, these phenacyl esters had identical relative extinction coefficients at 210, 240, and 280 nm. Phenacyl and methyl esters of Acet2 prepared from the ELI12-expressing soybean seeds were identical by 1H NMR spectroscopy to the corresponding derivatives of dehydrocrepenynic acid from the chanterelle mushrooms and to a chemically synthesized dehydrocrepenynic acid standard. A diagnostic vinyl resonance with a chemical shift (δ5.80) and coupling constants (dm, J = 10.7, 7.3 Hz), consistent with the presence of a cis-1,3-enyne in the dehydrocrepenynic ester, was observed. An additional diagnostic peak at 3.05 p.p.m., assigned to the skipped methylene protons in the 1,4-enyne system (C-11), was clearly visible in each sample. This resonance was down field compared to the chemical shift of the related polyalkenyl fatty acid resonances (e.g. methyl linoleate, 2.8 p.p.m.; Gunstone et al., 1977). Furthermore, the carbon-13 NMR data from methyl dehydrocrepenynate, prepared synthetically, from the previously published data (Vlahov, 1996), and from the acetylenase-expressing soybeans, were indistinguishable. The 1H NMR data are presented below in the following form: chemical shift in p.p.m. (multiplicity, coupling constant(s) in Hz and relative integration). Chemical shifts for proton-decoupled 13C NMR data (13C{1H}) are presented for the methyl ester. Phenacyl 14-cis-dehydrocrepenynate: 1H δ(CDCl3) 0.90 (t, J = 7.4, 3H), 1.2–1.5 (m, 10H), 1.69 (m, 2H), 2.03 (m, 2H), 2.24 (dq, J = 0.5, 7.4, 2H), 2.44 (t, J = 7.5, 2H), 3.06 (m, 2H), 5.32 (s, 2H), 5.38–5.48 (m, 3H), 5.80 (dt, J = 10.7, 7.4, 1H), 7.47 (m, 2H), 7.59 (m, 1H), 7.89 (m, 2H). Methyl 14-cis-dehydrocrepenynate: 1H δ(CDCl3) 0.90 (t, J = 7.4, 3H), 1.2–1.5 (m, 10H), 1.61 (m, 2H), 2.02 (m, 2H), 2.26 (m, 4H), 3.06 (m, 2H), 3.64 (s, 3H), 5.39–5.49 (m, 3H), 5.80 (dt, J = 10.7, 7.4, 1H); 13C{1H} δ(CDCl3) 174.29, 142.68, 131.64, 124.32, 109.34, 92.38, 77.20, 34.09, 32.07, 29.27, 29.10, 29.08, 29.03, 27.11, 24.92, 22.73, 22.14, 17.95, 13.73.

Overall, the production of the fatty acids containing Δ12 acetylenic or triple bonds in the transgenic soybean seeds expressing ELI12 thus demonstrates that this gene encodes a fatty acid acetylenase.

Identification of the fatty acid acetylenases in Araliaceae and Asteraceae species

During the course of our studies with ELI12, an EST was identified for a divergent form of FAD2 from the developing seeds of English ivy (Hedera helix), an Araliaceae species. ESTs for a class of divergent FAD2 were also identified among a cDNA population from the flower heads of sunflower (H. annuus), an Asteraceae species. The sunflower floral tissue used for the construction of this cDNA library contained developing seeds, and was collected from plants that had been infected with the fungal pathogen Sclerotinia sclerotiorum. Interestingly, six ESTs for the divergent FAD2 were identified among the 3153 total random cDNAs that were sequenced from this library (or 0.2% of the total ESTs). No ESTs for the typical or non-divergent class of FAD2 were found among this population of ESTs.

The divergent English ivy FAD2 corresponding to the EST shares 90% amino acid sequence identity with the parsley ELI12 polypeptide but <65% identity with all other reported FAD2 and divergent FAD2 gene products. In addition, phylogenetic comparisons shown in Figure 4 clearly indicate that the FAD2-like sequence from the English ivy is most related to the parsley ELI12 protein. Given the high degree of relation with the ELI12 polypeptide, it was expected that the English ivy enzyme is also a fatty acid acetylenase. Consistent with this, the expression of this polypeptide in soybean somatic embryos was accompanied by the accumulation of crepenynic and dehydrocrepenynic acids (Table 2). These two fatty acids together accounted for more than 6% of the total fatty acids of transgenic soybean embryos. Neither acetylenic fatty acid was detected in the non-transformed embryos.

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Figure 4. Phylogenetic comparison of the ELI12 polypeptide (PcACET) and divergent FAD2s from English ivy (HhACET), sunflower (HaACET), and Calendula officinalis (CoACET) characterized in this study with FAD2 and FAD2-related enzymes.

The unrooted phylogenetic tree shown was generated by use of the neighbor-joining algorithm (Saitou and Nei, 1987). Included in the alignment were FAD2 desaturases (DES), hydroxylases (OH), epoxygenases (EPOX), acetylenases (ACET), and conjugases (CONJ). The GenBank accession numbers of the amino acid sequences represented in the phylogenetic tree are: RcOH, T09839; AhDES, AAB84262; BoDES, AAC31698; GhDES, T10789; GmDES, P48630; CpEPOX, CAA76156; CaACET, CAA76158; CoACET, CAB64256; HaACET, AY166773; HhACET, AY166772; PcACET, U86734; CoCONJ, AF310155; IbCONJ, AAF05915; McCONJ, AAF05916; AtDES, P46313; LfOH, AAC32755; CpDES, CAA76157; and HaDES, T14269. (Pc, Petroselinum crispum; Rc, Ricinus communis; Ah, Arachis hypogea; Gm, Glycine max; Co, Calendula officinalis; Ib, Impatiens balsamina; Mc, Momordica charantia; At, Arabidopsis thaliana; Lf, Lesquerella fendleri; Bo, Borago officinalis; Gh, Gossypium hirsutum; Cp, Crepis palaestina; Ca, Crepis alpina; Ha, Helianthus annuus; Hh, Hedera helix).

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Table 2.  Fatty acid composition of somatic soybean embryos of a non-transformed line and transgenic lines expressing cDNAs for the English ivy (Hedera helix), sunflower (Helianthus annuus), or calendula (Calendula officinalis) acetylenases
Fatty acidWeight percentage of the total fatty acids
Non-transformed (n = 5)+English ivy acetylenase (n = 5)+Sunflower acetylenase (n = 5)+Calendula acetylenase (n = 6)
  • Values are presented as weight percentage of the total fatty acids of embryos and were obtained from analyses of 5–6 single embryos (±SD) from each line.

  • 1

    N.D., Not detected.

  • 2

    Includes 20:0 and 22:0.

16:016.1 ± 0.917.4 ± 0.914.5 ± 1.114.5 ± 0.7
18:02.0 ± 0.12.2 ± 0.22.8 ± 0.43.4 ± 0.7
18:19.4 ± 1.210.4 ± 0.910.4 ± 1.19.5 ± 2.1
18:252.5 ± 1.248.8 ± 4.353.7 ± 2.050.8 ± 2.1
18:318.5 ± 1.914.0 ± 3.516.1 ± 1.719.1 ± 3.6
CrepenynicN.D.12.2 ± 0.30.7 ± 0.20.8 ± 0.1
DehydrocrepenynicN.D.3.9 ± 0.21.0 ± 0.20.8 ± 0.1
Other2≤1.4≤1.0≤1.3≤1.0

In contrast to the English ivy acetylenase, the divergent sunflower FAD2 deduced from the ESTs shares only 57% amino acid sequence identity with the ELI12 polypeptide. In addition, phylogenetic comparisons of the divergent sunflower FAD2 with other FAD2 and FAD2-related polypeptides indicated that this enzyme is evolutionarily distinct from either the English ivy acetylenase or the parsley ELI12 enzyme (Figure 4). Instead, the divergent sunflower FAD2 is most related too with fatty acid acetylenases and epoxygenases from seeds of Crepis species (Lee et al., 1998). The sunflower polypeptide shares approximately 75% amino acid sequence identity with the Crepis FAD2-related enzymes. Interestingly, the sunflower polypeptide also shares 91% identity with a divergent FAD2 from pot marigold (Calendula officinalis), which was previously identified, based on limited analytical evidence, as a Δ8, Δ11-desaturase that forms conjugated double bonds (Fritsche et al., 1999). To clarify its function, the divergent sunflower FAD2 was expressed in the soybean somatic embryos. Accompanying the expression of this enzyme was the production of crepenynic and dehydrocrepenynic acids, which accumulated to nearly 2% of the total fatty acids of the transgenic embryos. No other unusual fatty acids were detected in these embryos (Table 2). This result thus indicated that the divergent sunflower FAD2 functions as a fatty acid acetylenase.

This result prompted us to re-assess the function of the C. officinalis‘Δ8, Δ11 desaturase’ (Fritsche et al., 1999), given its high degree of identity with the sunflower fatty acid acetylenase described above. The ascribed identity of the Calendula enzyme was based on ultraviolet analysis of the fatty acids obtained from the expression of the enzyme in S. cerevisiae. However, no GC or GC–MS data were provided to support this functional identification. More recently, two independent reports have conclusively identified a different and more divergent form of FAD2 from C. officinalis as the true Δ8, Δ11-desaturase that is associated with the formation of the fatty acids with conjugated double bonds in seeds of this plant (Cahoon et al., 2001; Qiu et al., 2001). To provide more conclusive evidence of its function, the C. officinalis FAD2 with high identity to the sunflower acetylenase was expressed in the somatic soybean embryos. Accompanying the expression of this enzyme was the accumulation of crepenynic and dehydrocrepenynic acids comprising 1–2% of the total fatty acids of the transgenic embryos (Table 2). Based on this finding, it is likely that the divergent C. officinalis FAD2 was previously misidentified by Fritsche et al. (1999), and that instead it functions as a fatty acid acetylenase.

The results described above thus demonstrate that the FAD2-related fatty acid acetylenases are expressed not only in the Apiaceae species (e.g. parsley), but also in the members of the Araliaceae (e.g. English ivy) and Asteraceae (e.g. sunflower and C. officinalis) families.

Occurrence of FAD2-related fatty acetylenase genes in other Apiaceae and Asteraceae species

To provide evidence for a general occurrence of fatty acid acetylenase genes in Apiaceae and Asteraceae species, PCR amplification was conducted with degenerate oligonucleotide primers that correspond to partially conserved domains in FAD2 and FAD2-related genes. The template for these reactions was genomic DNA isolated from a variety of Apiaceae and Asteraceae species. Of note, all previously characterized FAD2 and FAD2-related genes lack introns within their open-reading frames (Cahoon, unpublished observation). Thus, the PCR-based analysis of the genomic DNA provides a straightforward and widely applicable tool for characterization of the FAD2-type genes in plants.

Among the PCR products obtained from the genomic DNA of the Asteraceae black-eyed Susan (Rudbeckia hirta), strawflower (Helichrysum bracteatum) and cape daisy (Dimorphotheca sinuata) were sequences encoding a FAD2-related class that displayed a high degree of homology with the sunflower FAD2 acetylenase described above. The polypeptide sequences deduced from the R. hirta, H. bracteatum, and D. sinuata PCR products shared 94, 89, and 89% identity, respectively, with the sunflower acetylenase. In addition, phylogenetic analysis indicated that these polypeptides are members of the Asteraceae class of fatty acid acetylenases and epoxygenases (Figure 5). As the R. hirta, H. bracteatum, and D. sinuata enzymes are most related to the sunflower and C. officinalis fatty acid acetylenases, they are predicted to have the same function.

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Figure 5. Full-length sequences were compared with sequence fragments obtained by PCR from genomic DNA of Rudbeckia hirta (RhFAD2-PCR), Helichrysum bracteatum (HbFAD2-PCR), Dimorphotheca sinuata (DsFAD2-PCR), Daucus carota (DcFAD2-PCR), and Foeniculum vulgare (FvFAD2-PCR). The FAD2 and FAD2-related polypeptides and algorithm that were used for generation of the dendrogram are described in the legend of Figure 4.

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In addition, the PCR products obtained from the genomic analysis of the Apiaceae species, carrot (Daucus carota) and fennel (Foeniculum vulgare), included a class of sequences that encode polypeptides with 91% amino acid sequence identity with the parsley ELI12 polypeptide and 88–90% identity with the English ivy acetylenase. In contrast, these polypeptides share <65% amino acid sequence identity with all the previously reported FAD2 and FAD2-related polypeptides. The polypeptides deduced from the D. carota and F. vulgare PCR products, thus, clearly group with the Apiaceae and Araliaceae class of fatty acid acetylenases in phylogenetic comparisons (Figure 5), and likely catalyze the same chemical processes.

These results suggest that genes for FAD2-related fatty acid acetylenases are not limited to parsley within the Apiaceae family or to sunflower and C. officinalis in the Asteraceae family, but occur more widely in these families.

Sclerotinia induction of fatty acid acetylenase gene expression in sunflower

It is known that expression of the parsley ELI12 is induced by fungal elicitation of the cell suspension cultures (Somssich et al., 1989). In our work, Northern blot analysis was conducted to determine whether an Asteraceae-type fatty acid acetylenase gene can be upregulated in response to fungal infection in plant tissue. In these experiments, sunflower roots, petioles, and flower heads were inoculated with the fungal pathogen S. sclerotiorum. Tissues from the infected plants were then analyzed for expression of the fatty acetylenase gene. Expression of the fatty acid acetylenase gene was detected primarily in the developing seeds, and this expression was induced by infection of flower heads with S. sclerotiorum (Figure 6).

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Figure 6. Expression of the FAD2 acetylenase gene in tissues of uninfected sunflower (–) plants or plants infected with the fungal pathogen Sclerotinia sclerotiorum (+).

Total RNA (10 µg) isolated from the sunflower tissues was electrophoresed, blotted, and hybridized with a 32P-radiolabeled probe derived from the 3′-non-translated portion of the sunflower acetylenase cDNA. Infected stem and leaf were obtained from plants inoculated by treatment of petioles with fungal mycelia. Infected receptacle, corolla, and developing seeds were obtained from plants inoculated by treatment of flower heads with fungal ascospores. Infected roots were obtained from plants treated with an oat-based fungal inoculum as previously described by Mancl and Shein (1982). The ethidium bromide-stained gel corresponding to the Northern blot is shown in the lower panel.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The fungal elicitor-induced ELI12 gene from parsley has previously been shown to encode a divergent form of the Δ12-oleic acid desaturase or FAD2 (Kirsch et al., 1997a). Previous attempts to identify the function of the ELI12 gene product, however, have not been successful (Kirsch et al., 1997a). Here, we show that the expression of this gene in transgenic soybean seeds results in the accumulation of acetylenic fatty acids. The in vivo activity of the ELI12 gene product is thus consistent with that of a fatty acid acetylenase. A FAD2-related enzyme of similar activity has been previously identified in seeds of Crepis alpina, which accumulate large amounts of the acetylenic fatty acid, crepenynic acid (Lee et al., 1998). Parsley, however, is not known to accumulate detectable amounts of acetylenic fatty acids in any of its tissues, but has been reported to produce other acetylenic compounds that are derived from fatty acid precursors (Bohlmann, 1967). In this study, we have also shown that fatty acid acetylenases occur not only in the Apiaceae species, such as parsley, but also in members of the Araliaceae and Asteraceae families, and expression of the acetylenase gene from the Asteraceae sunflower, like ELI12, is induced by fungal elicitation. Interestingly, the Apiaceae and Araliaceae acetylenases share approximately 90% amino acid sequence identity; however, Asteraceae acetylenases share <60% identity with those from Apiaceae and Araliaceae species. This finding, as indicated by the phylogenetic comparisons in Figures 4 and 5, suggests that fatty acid acetylenases in Apiaceae and Araliaceae arose independently from acetylenases in Asteraceae.

Recently, a second class of divergent FAD2 genes has been identified in parsley. This class, which is designated as ELI7, contains at least two closely related and genetically linked genes (ELI7.1 and ELI7.2; Kirsch et al., 2000). Like ELI12, these genes are also induced by fungal elicitation (Kirsch et al., 2000). The ELI7.1 and ELI7.2 polypeptides share 90–92% amino acid sequence identity with the ELI12-encoded acetylenase and 88% identity with the English ivy enzyme. Given this high degree of amino acid sequence identity, it is likely that the ELI7 gene products also function as fatty acid acetylenases.

In addition to our demonstration of the FAD2-related acetylenases in the Apiaceae, Araliaceae, and Asteraceae families, a viral-induced divergent FAD2 has been identified in the Solanaceae species tomato (Gadea et al., 1996). ESTs for a closely related FAD2 have also been identified from potato leaves that were infected with the fungal pathogen Phytophthora infestans (GenBank accession no. BG591451). These divergent FAD2s from Solanaceae species share 55–65% identity with all the previously reported FAD2 and the functionally divergent FAD2 polypeptides. The activity of these enzymes, however, is not evident from phylogenetic comparisons (data not shown). Regardless of this, the identification of cDNAs for these enzymes in Solanaceae species demonstrates that the occurrence of pathogen-induced FAD2-like polypeptides extends beyond the Apiaceae, Araliaceae, and Asteraceae families.

One notable aspect of our results is the detection of dehydrocrepenynic acid (12-acetylenic-18:2Δ9cis,14cis) in soybean seeds and somatic embryos upon expression of fatty acid acetylenases from the Apiaceae, Araliaceae, and Asteraceae species. This fatty acid has been previously identified as a major component of seed oil of the tropical legume genera Afzelia (Gunstone et al., 1972) and of mushrooms of the genus Cantharellus (Pang and Sterner, 1991). The biosynthetic origin of the conjugated Δ14 double bond in dehydrocrepenynic acid, rather than the more typical non-conjugated Δ15 double bond, is not clear. One possibility is that the triple bond of crepenynic acid results in an altered substrate conformation in the active site of the Δ15-desaturase (FAD3) such that the double bond is inserted at the Δ14 position. Another possibility is that the acetylenases described here are able to function on α-linolenic acid (18:3Δ9,12,15) in addition to linoleic acid (18:2Δ9,12). In such a metabolic scenario, introduction of the Δ12 triple bond is accompanied by the re-arrangement of the Δ15 double bond of α-linolenic acid to the Δ14 position. Our preliminary attempts to experimentally distinguish between these and other possible pathways were complicated by the relatively low accumulation of dehydrocrepenynic acid in the transgenic tissue.

The major question that arises from our results is the biological significance of the fatty acid acetylenases in the Apiaceae, Araliaceae, and Asteraceae species. Most plants of these families, including parsley, English ivy, and sunflower, are not known to accumulate the crepenynic and dehydrocrepenynic acids, which are the acetylenic fatty acids that arise from the transgenic expression of these enzymes (as described in this study). However, members of the Apiaceae, Araliaceae, and Asteraceae families have been shown to produce linear polyacetylenic compounds, many of which have demonstrated biological activity. These compounds include falcarinol (also known as carotatoxin and panaxynol) and falcarindiol in Apiaceae and Araliaceae species, and dehydrofalcarinol in Asteraceae species (Bohlmann et al., 1973; Figure 7). Interestingly, the occurrence of polyacetylenic compounds, including falcarinol and falcarindiol, has also been reported in plants of the Solanaceae family (Imoto and Ohta, 1988; deWit and Kodde, 1981). In fact, polyacetylenic compounds have been detected in at least 15 families, including those described here (Bohlmann et al., 1973). As shown in Figure 7, crepenynic and dehydrocrepenynic acids are believed to be intermediates in the biosynthetic pathway of these polyacetylenes. Consistent with this pathway, 14C-labeled crepenynic acid has been shown to be incorporated into falcarinol when provided as an exogenous precursor to carrot cells (Barley et al., 1988). Similarly, various metabolic studies have implicated crepenynic and dehydrocrepenynic acids as intermediates in the synthesis of related polyacetylenes produced by fungi such as Tricholoma grammopodium (Bu'Lock and Smith, 1967; Farrell et al., 1987). Of note, the proposed polyacetylene pathway shown in Figure 7 infers the existence of several as yet unidentified enzymes including a second acetylenase that modifies the Δ14 double bond of dehydrocrepenynic acid.

image

Figure 7. Proposed biosynthetic pathway for selected Asteraceae, Apiaceae, and Araliaceae acetylenic natural products.

As indicated, the initial step in the proposed pathway is catalyzed by a Δ12-fatty acid acetylenase. Subsequent steps in the pathway are based upon previously reported radioactive tracer experiments (Barley et al., 1988; Bohlmann et al., 1973; Farrell et al., 1987). It is not known whether fatty acid intermediates are unesterified or are bound to other molecules (e.g. R may be H or a glycerolipid). However, it is likely that linoleic acid in the initial reaction is bound to a phospholipid, particularly phosphatidylcholine (PC), given the homology of the acetylenases to the acyl-PC-requiring FAD2 enzymes. Formation of the terminal alkene may be mediated by hydroxylase/elimination sequence (Benveniste et al., 1998).

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Numerous biological activities have been attributed to the polyacetylenes produced by the Apiaceae, Araliaceae, and Asteraceae species. Falcarindiol, for example, is a potent antifungal agent (Garrod and Lewis, 1979; Villegas et al., 1988), and is believed to provide a defense against fungal pathogenesis of Apiaceae species such as carrot (Kemp, 1978; Olsson and Svensson, 1996). This compound has also demonstrated insecticidal (Eckenbach et al., 1999), antimicrobial (Muir and Majak, 1983), and antiproliferative activities (Nakano et al., 1998). Falcarinol is a potent inhibitor of fatty acid oxygenases including lipoxygenases and cyclooxygenases (Alanko et al., 1994). This acetylenic alcohol, which accumulates together with its derivatives in the leaves and stems of many members of Araliaceae (Boll and Hansen, 1987), is the primary allergen associated with the contact-dermatitis response of English ivy (Gafner et al., 1989). This compound also inhibits seed germination, and may contribute to the nuisance spread of English ivy (Nitz et al., 1990). In addition to the polyacetylenes shown in Figure 7, numerous other bioactive acetylenic compounds have been reported in plants. These include acetylenic thiophenes such as those associated with the insecticidal and nematicidal properties of Tagetes species (e.g. marigold) (Jacobs et al., 1995; Towers et al., 1997).

In fungal elicitor-treated parsley leaves, we were able to measure induction of ELI12 gene expression, but were unable to detect the accumulation of crepenynic or dehydrocrepenynic acid in the total lipid extract. In addition, although we did not analyze the fatty acid content of the sunflower seeds from the Sclerotinia-infected flowers, the acetylenic fatty acids are not known to accumulate in seed oils of most of the members of the Asteraceae family, including sunflower. Based on these observations, it is likely that acetylenic fatty acids are synthesized in low amounts and/or are rapidly metabolized for the formation of secondary bioactive molecules such as falcarinol in response to fungal pathogenesis. Additionally, the high levels of crepenynic acid that occur in seed oils of the Asteraceae species such as C. alpina may have arisen from a lost or reduced ability to metabolize acetylenic fatty acids into polyacetylenes.

Results of metabolic studies to date are consistent with the role of crepenynic and dehydrocrepenynic acids as precursors of polyacetylenes that are known to occur in Apiaceae, Araliaceae, and Asteraceae as well as other families such as the Solanaceae (Bohlmann et al., 1973). The fatty acid acetylenases described in this report are strongly implicated as components of the secondary metabolic pathways that give rise to these biologically active compounds. The divergent FAD2 cDNAs and genes that we have characterized in this report will likely provide molecular tools to manipulate amounts of polyacetylenes to assess their contribution to the natural defense of plants against pathogens including fungi, bacteria, nematodes, and insects.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Isolation and transgenic expression of the ELI12 open-reading frame

The open-reading frame for the ELI12 gene was amplified by PCR from genomic DNA isolated from the leaves of Italian parsley (P. crispum cv. Neapolitanum) as previously described by Shure et al. (1983). (Note: FAD2 and divergent FAD2 genes typically do not contain introns within their open-reading frames (Cahoon, unpublished result)). The oligonucleotide primers used to amplify the ELI12 open-reading frame were: 5′-TTAATGCGGCCGCAAGTATGGGTGCAGGTGGACG-3′ (sense) and 5′-TTAATGCGGCCGCTTAAAACTTGTTTCTGTACCAGTAG-3′ (antisense). These primers were designed based on the previously reported ELI12 sequence (GenBank accession no. U86734; Kirsch et al., 1997a). Three independent PCR reactions were conducted using the Pfu polymerase (Stratagene, La Jolla, CA, USA), and the sequences of the subcloned products from each reaction were found to be identical. The amplified sequences encoded a polypeptide that differed from the published ELI12 sequence (Kirsch et al., 1997a) by three amino acids over a total of 383 residues. The amino acid differences between our sequence and the published sequence are G239D, I270Y, and A333S. In the case of the latter two amino acids, the Ile and the Ala residues deduced from our sequence are perfectly conserved in all previously reported FAD2 and divergent FAD2s. This suggests that residues 270 and 333 are incorrect in the published ELI12 amino acid sequence.

The amplified ELI12 open-reading frame was introduced as a NotI restriction fragment into the soybean expression vector pKS67 (Cahoon et al., 1999). The resulting plasmid (designated as pFEI-F2) consisted of the ELI12 open-reading frame linked at its 5′ end to the promoter of the gene for the α′-subunit of β-conglycinin (Doyle et al., 1986), which directs strong seed-specific expression of transgenes. Selection of the transgenic plant cells in pFEI-F2 was conferred by a hygromycin B phosphotransferase gene (Gritz and Davies, 1983) linked to the cauliflower mosaic virus 35S promoter.

Transgenic soybean (Glycine max cv. Jack) plants were generated by particle bombardment of the somatic embryos with pFEI-F2, using previously described methods (Cahoon et al., 1999; Jung and Kinney, 2001). Expression of the ELI12 open-reading frame was confirmed by Northern blot analysis of seeds from plants regenerated from hygromycin-selected embryos as described previously (Cahoon et al., 2001). Results reported were obtained from the homozygous seeds of the transgenic event MSP147-1-4-4.

Expression of cDNAs for divergent FAD2 ESTs in somatic soybean embryos

Expressed sequence tag analysis was conducted, as previously described (Cahoon et al., 1999, 2001), with cDNA libraries prepared from developing English ivy (H. helix) seeds and flower heads (containing developing seeds) of Sclerotinia-infected sunflower (H. annuus). From these analyses, full-length cDNAs for divergent forms of FAD2 were identified. The English ivy (GenBank accession no. AY166772) and sunflower (GenBank accession no. AY166773) divergent FAD2 cDNAs were expressed in soybean somatic embryos to determine the function of the corresponding polypeptides. To facilitate cloning into the soybean expression vector, the English ivy cDNA was amplified by PCR with the following primers: 5′-TTGCGGCCGCTGCACATCAAAGAAAAATGG-3′ (sense) and 5′-TTGCGGCCGCAGCATGCACATCAAAACTCAC-3′ (antisense). Oligonucleotide primers used for amplification from the sunflower cDNA were: 5′-ATGCGGCCGCTCAAACATCCACCAACATGG-3′ (sense) and 5′-ATGCGGCCGCGTACACTGATTACATTTTATG-3′ (antisense). Pfu polymerase (Stratagene, La Jolla, CA, USA) was used for PCR amplification of both cDNAs. The products of the PCR reactions were introduced as NotI restriction fragments into the soybean expression vector pKS67, as described. The resulting plasmids containing the divergent FAD2 cDNAs linked to the promoter for the α′-subunit of the β-conglycinin gene were stably transformed into the somatic soybean embryos, as described. The fatty acid composition was then determined for transgenic embryos selected with hygromycin and maintained as described by Cahoon et al. (1999). The results presented were from transformation events MSE 342-9-1 (divergent English ivy FAD2) and MSE 354-5-18 (divergent sunflower FAD2). Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes.

Transgenic expression of a divergent FAD2 from Calendula officinalis in somatic soybean embryos

A divergent FAD2 from C. officinalis (GenBank accession no. AJ245938) was expressed in soybean somatic embryos in order to re-assess the previous identification of its gene product as a ‘(8,11)-linoleoyl desaturase’ (Fritsche et al., 1999). The open-reading frame of the C. officinalis gene was amplified by PCR from genomic DNA isolated from the leaves of C. officinalis. Pfu polymerase was used in the PCR reaction along with the following primers: 5′-ATTGCGGCCGCATGGGTGCTGGTGGTCGGATGTCG-3′ (sense) and 5′-TTTGCGGCCGCTGACATACACCTTTTTGATTACATC-3′ (antisense). Both the strands of the amplification product were sequenced prior to its insertion as a NotI fragment into the vector pKS67 (described earlier). The resulting plasmid was used for transformation of the somatic soybean embryos using the detailed methodology (Jung and Kinney, 2001). Expression of the divergent C. officinalis FAD2 in soybean somatic embryos was confirmed by PCR with sequence-specific primers and template consisting of first-strand cDNA synthesized from RNA isolated from the transgenic embryos. RNA was extracted from the soybean embryos with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and first strand cDNA was synthesized from the total RNA using an oligo-dT primer and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocols. Results reported were from transgenic event MSE 585-1-8.

Fatty acid analysis of transgenic soybean seeds and somatic embryos

Fatty acid methyl esters (FAMEs) were prepared from non-transformed soybean seeds or seeds expressing ELI12 by direct transesterification of the chipped portions (approximately 5–10 mg dry weight) of individual mature seeds in trimethylsulfonium hydroxide (TMSH)/methanol (Butte et al., 1982). Seed chips were obtained from the side opposite of the hilum using a razor blade, and were placed in a gas chromatograph (GC) autosampler vial. Heptane (500 µl) and TMSH reagent (50 µl) were then added to each vial. Capped vials were then incubated at room temperature with vigorous shaking for 20 min prior to GC analysis.

Fatty acid methyl esters were prepared from soybean somatic embryos by direct transesterification of single embryos in 1% (w/w) sodium methoxide in methanol using methods identical to those previously described (Cahoon et al., 1999).

The composition of FAMEs prepared from seeds or somatic embryos was determined with a Hewlett-Packard 6890 GC (Palo Alto, CA, USA) fitted with an Omegawax 320 column (30 m × 0.32 mm inner diameter; Supelco, Bellefonte, PA, USA). The oven temperature was programmed from 200°C (2.6 min hold) to 240°C at a rate of 20°C min−1, and FAMEs were measured by flame-ionization detection. To provide further structural characterization, FAMEs were analyzed by gas chromatography–mass spectrometry (GC–MS) using a Hewlett Packard 6890 GC coupled with a Hewlett Packard 5973 mass selective detector. Fatty acid methyl esters from the soybean seeds and the somatic embryos were separated with an INNOWax column (30 m × 0.25 mm inner diameter; Agilent, Palo Alto, CA, USA). The oven temperature was programmed from 185°C (3.5 min hold) to 215°C (5 min hold) at a rate of 2°C min−1, and then to 230°C at a rate of 5°C min−1. Fatty acid methyl esters were identified by the retention times and the mass spectral fragmentation patterns relative to commercial standards or methyl esters of crepenynic and dehydrocrepenynic acids purified from natural sources (see below). Acetylenic FAMEs from ELI12-transformed soybeans were also compared to synthetic methyl crepenynate as well as to the 14-cis isomer of methyl dehydrocrepenynate from both natural and synthetic origins by GC retention time, mass spectral fragmentation pattern, and GC co-injection experiments. The synthesis of methyl 14-cis-dehydrocrepenynate has been previously reported by Zhu and Minto (2001).

Purification of methyl esters of crepenynic and dehydrocrepenynic acids from natural sources

Methyl esters of crepenynic and dehydrocrepenynic acids for GC and GC–MS standards were prepared and isolated from extracts of C. rubra seeds and chanterelle mushrooms. Crepenynic acid typically composes >60 wt.% of the total fatty acids of C. rubra seeds (Mikolajczak et al., 1964), and dehydrocrepenynic acid typically composes of >25% of the total fatty acids of chanterelle mushrooms (Pang and Sterner, 1991).

Methyl crepenynate was prepared by initially heating 100 mg aliquots of C. rubra seeds in 2.5% (v/v) sulfuric acid/methanol (2 ml) at 80°C for 1 h. The resulting FAMEs were extracted with heptane following the addition of saturated NaCl solution (2 ml) to the cooled reaction. FAMEs were then resolved by thin layer chromatography (TLC) on C18 reverse-phase plates (KC18 plates, 200 µm layer, 10 cm × 20 cm; Whatman, Clifton, NJ, USA), using a mobile phase of methanol:acetonitrile:water (60:40:1, v/v/v). To improve resolution, the TLC plates were developed sequentially with the same mobile phase to one-third, two-thirds, and the full-length of the plate. The plates were dried in a nitrogen atmosphere between the three developments. The methyl crepenynate was identified by its slightly higher mobility relative to the methyl linolenate. The methyl crepenynate was visualized by light staining with iodine vapors and then recovered from the TLC plate scrapings using hexane:isopropanol (7:2, v/v). After drying under nitrogen, the purified sample was re-suspended in hexane:ethyl ether (80:20, v/v) and applied to a 1-cm column of silica gel prepared in a Pasteur pipet, and was equilibrated in the hexane:ethyl ether solution. The methyl crepenynate was then eluted with 5 ml of hexane:ethyl ether (80:20, v/v).

The methyl ester of dehydrocrepenynic acid was prepared from dried chanterelle mushrooms purchased at a local grocery store. The mushrooms were initially pulverized to a fine powder with a mortar and pestle. The powdered mushrooms were heated for 40 min in 2.5% (v/v) sulfuric acid/methanol (2 ml) at 80°C for 1 h. The resulting FAMEs were extracted with heptane following the addition of saturated NaCl solution (2 ml) to the cooled reaction. Methyl dehydrocrepenynate was purified from other fatty acid methyl esters in the extract using the TLC protocol described above for the purification of methyl crepenynate.

Derivatization and HPLC purification of acetylenase products

Five finely ground ELI12-transformed soybean seeds were extracted with CHCl3:MeOH (2:1 v/v) by stirring for 90 min. The extraction solvent was collected by filtration and concentrated to dryness using a rotary evaporator.

Crude lipids were also extracted from pulverized, dried C. cibarius fruit bodies (24 g). The tissue was finely pulverized in a coffee mill, was transferred to a round-bottom flask, and was stirred in ethyl acetate (300 ml) for 5 h. The liquid was isolated and concentrated to an oil under vacuum at room temperature.

Soybean- and chanterelle-derived lipids and synthetic methyl dehydrocrepenynate were saponified with 0.5% (w/v) methanolic sodium hydroxide by heating in a water bath at 100°C for 5 min, cooling to room temperature, and repeating the heating step. The solutions were acidified with concentrated HCl, and the fatty acids were extracted twice with hexane. The hexane was evaporated in vacuo, the free fatty acids were dissolved in acetone (13 ml), and triethylamine (180 µl) was added. The solution was mixed prior to the addition of phenacyl bromide (156 mg). The reaction mixture was heated at 50°C for 2 h. Water (5 ml) was added and the solution was extracted twice with hexane. Concentration of the organic phase yielded the phenacyl derivatives. Phenacyl derivatives of commercially available fatty acids were prepared as chromatography standards.

Fatty acid phenacyl derivatives were separated on an Econosil C18 column (250 mm × 10 mm internal diameter; Supelco, Bellefonte, PA, USA). Separation of phenacyl derivatives was performed at a flow rate of 5 ml min−1 at 4°C using a linear gradient from 10% water/90% acetonitrile (v/v) to 100% acetonitrile over 40 min. The mobile phase was then maintained at 100% acetonitrile for 40 min. The elution was monitored at 210, 240, and 280 nm. Under these conditions, phenacyl dehydrocrepenynate from chanterelle extracts and synthetic sources was observed to elute at 68 ml. A phenacyl derivative with identical extinction coefficients at the monitored wavelengths was purified from extracts of soybeans expressing ELI12 by collection of the 68 ml eluate.

Proton (300 MHz) and 13C{1H} (75.5 MHz) NMR spectroscopy of phenacyl and methyl ester derivatives of dehydrocrepenynate from biological (transgenic soybeans and chanterelle mushrooms) and synthetic sources were performed on a Bruker Avance spectrometer (Billerica, MA, USA). Spectra were recorded at 298 K in CDCl3. Chemical shifts in p.p.m. were solvent-referenced and expressed down field from tetramethylsilane.

PCR-based analysis of FAD2 acetylenase-like genes in Asteraceae and Apiaceae

The occurrence of FAD2 acetylenase-like genes in other Asteraceae and Apiaceae species was detected by PCR amplification from genomic DNA using degenerate oligonucleotide primers designed from partially conserved amino acid sequences in FAD2 and divergent FAD2 polypeptides, including FAD2-related acetylenases. Genomic DNA was isolated from leaves of the Asteraceae species R. hirta, H. bracteatum, and D. sinuata using the previously described method (Shure et al., 1983). Genomic DNA was also isolated from leaves of the Apiaceae species D. carota and F. vulgare, using the previously described method (Asemota, 1995). The degenerate oligonucleotide primers used for amplification from the genomic DNA of Asteraceae species were: 5′-tagagctcAAIAARGCNATHCCNCCNCAYTGYTT-3′ (sense), which corresponds to the amino acid sequence KKAIPPHCF, and 5′-taagatctgtatacRCAYTCYTTNGCYTCNCKC-3′ (antisense), which corresponds to the amino acid sequence REAKEC. The sense primer used for amplification from the genomic DNA of Apiaceae species was the same as described for Asteraceae species, and the antisense primer for Apiaceae species was 5′-atgaattcCYTTCATYTCNCKRTACATNGC-3′, which corresponds to the amino acid sequence AMYREMK. (Note: Sequences shown in lower case contain restriction sites and additional nucleotides to facilitate restriction enzyme digestion.) This amino acid sequence is found in the ELI12- and the divergent English ivy FAD2 polypeptides, but is only partially conserved in other FAD2 and divergent FAD2 polypeptides. Forty cycles of PCR amplification were conducted using Taq polymerase in a 100 µl reaction volume that contained 150 ng of genomic DNA from the Asteraceae and Apiaceae species. The annealing temperature was 50°C. The resulting products of approximately 1 kb were subcloned into pGEM-T (Promega, Madison, WI, USA), according to the manufacturer's protocol, and transformed into Escherichia coli DH10B cells (Gibco-BRL, Carlsbad, CA, USA). Nucleotide sequences were then obtained for cDNA inserts from plasmids of the resulting colonies. GenBank accession numbers for the PCR-derived sequences from R. hirta, H. bracteatum, D. sinuata, D. carota, and F. vulgare are AY166776, AY166778, AY16777, AY166774, and AY166775, respectively.

Northern blot analysis of Sclerotinia-infected sunflower

Sunflower plants were infected with S. sclerotiorum by either spraying flower heads with ascospores, treatment of petioles with fungal mycelia, or treatment of roots with an oat-based innoculum as previously described (Mancl and Shein, 1982). Tissue samples were collected upon initial appearance of fungal infection symptoms (typically 7–20 days after inoculation). Receptacles, corolla, and developing seeds (approximately 14 days after pollination) were collected from the fungus-infected flower heads. Leaves and stems were collected from the petiole-infected plants.

Tissues from sunflower infected with S. sclerotiorum were ground into fine powder in liquid nitrogen, and total RNA was prepared using TriPure Reagent (Boehringer, Ingleheim, Germany), according to the manufacturer's protocol. Twenty micrograms of total RNA was separated in a 1% agarose gel containing formaldehyde. Ethidium bromide was included in the gel to verify equal loading of RNA. After transferring onto a Hybond N+ membrane (Amersham, Little Chalfont, UK), the blots were hybridized overnight at 65°C with a 32P-labeled probe. The template for the probe was generated from a purified PCR product amplified from the 3′-non-translated region of the sunflower FAD2 acetylenase cDNA using the following sense and antisense primers: 5′-CAAGAGACGCAATCAAGCC-3′ and 5′-CCACTTGTATATTTATGGAG-3′, respectively. Hybridization and washing conditions were performed as previously described (Church and Gilbert, 1984).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Zhongmeng Bao, Xiamomei Shi, and Guihua Lu for performing Northern blot analyses of sunflower, and Michael Parsons and Mark Mancl for preparing Sclerotinia-infected sunflower tissues. We also thank Maureen Dolan and associates at DuPont Genomics for EST sequencing of the cDNA libraries. We are also grateful to Sarah Hall for technical assistance and to George Cook, Bruce Schweiger, and Christine Howells for conducting soybean transformations. R.E.M. appreciates the research support provided by the state funds appropriated to the Ohio Plant Biotechnology Consortium through The Ohio State University, Ohio Agricultural Research and Development Center.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Alanko, J., Kurahashi, Y., Yoshimoto, T., Yamamoto, S. and Baba, K. (1994) Panaxynol, a polyacetylene compound isolated from oriental medicines, inhibits mammalian lipoxygenases. Biochem. Pharmacol. 48, 19791981.
  • Asemota, H.N. (1995) A fast, simple, and efficient mini-scale method for the preparation of DNA from tissues of yam (Dioscorea spp.). Plant Mol. Biol. Rep. 13, 214218.
  • Barley, G.C., Jones, E.H.R. and Thaller, V. (1988) Crepenynate as a Precursor of Falcarinol in Carrot Tissue Culture. Amsterdam: Elsevier.
  • Benveniste, I., Tijet, N., Adas, F., Philipps, G., Salaun, J.P. and Durst, F. (1998) CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase. Biochem. Biophys. Res. Commun. 243, 688693.
  • Bohlmann, F. (1967) Polyacetylene compounds. CXXXIX. Components of parsley and celery roots. Chem. Ber. 100, 34543456.
  • Bohlmann, F., Burkhardt, T. and Zdero, C. (1973) Naturally Occurring Acetylenes. London: Academic Press.
  • Boll, P.M. and Hansen, L. (1987) On the presence of falcarinol in Araliaceae. Phytochem. 26, 29552956.
  • Bu'Lock, J.D. and Smith, G.N. (1967) The origin of naturally-occurring acetylenes. J. Chem. Soc. (C). 1967, 332336.
  • Butte, W., Eilers, J. and Kirsch, M. (1982) Trialkylsulfonium and trialkylselenonium hydroxides for the pyrolytic alkylation of acidic compounds. Anal. Lett. 15, 841850.
  • Cahoon, E.B., Carlson, T.J., Ripp, K.G., Schweiger, B.J., Cook, G.A., Hall, S.E. and Kinney, A.J. (1999) Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos. Proc. Natl. Acad. Sci. USA, 96, 1293512940.
  • Cahoon, E.B., Ripp, K.G., Hall, S.E. and Kinney, A.J. (2001) Formation of conjugated Δ8, Δ10-double bonds by Δ12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid. J. Biol. Chem. 276, 26372643.
  • Church, G.M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81, 19911995.
  • Doyle, J.J., Schuler, M.A., Godette, W.D., Zenger, V., Beachy, R.N. and Slightom, J.L. (1986) The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. Structural homologies of genes and proteins. J. Biol. Chem. 261, 92289238.
  • Eckenbach, U., Lampman, R.L., Seigler, D.S., Ebinger, J. and Novak, R.J. (1999) Mosquitocidal activity of acetylenic compounds from Cryptotaenia canadensis. J. Chem. Ecol. 25, 18851893.
  • Farrell, I.W., Higham, C.A., Jones, E.R.H. and Thaller, V. (1987) Natural acetylenes. Part 62. Fungal polyacetylenes and the crepenynate pathway: experiments relevant to the biogenesis of the acetylene bond. J. Chem. Res. (S), 1987, 234235.
  • Fritsche, K., Hornung, E., Peitzsch, N., Renz, A. and Feussner, I. (1999) Isolation and characterization of a calendic acid producing (8,11)-linoleoyl desaturase. FEBS Lett. 462, 249253.
  • Gadea, J., Mayda, M.E., Conejero, V. and Vera, P. (1996) Characterization of defense-related genes ectopically in viroid-infected tomato plants. Mol. Plant–Microbe Interact. 9, 409415.
  • Gafner, F., Reynolds, G.W. and Rodriguez, E. (1989) The diacetylene 11,12-dehydrofalcarinol from Hedera helix. Phytochemistry, 28, 12561257.
  • Garrod, B. and Lewis, G.B. (1979) Location of the antifungal compound falcarindiol in carrot root tissue. Trans. Br. Mycol. Soc. 72, 515517.
  • Gritz, L. and Davies, J. (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene, 25, 179188.
  • Gunstone, F.D., Pollard, M.R., Scrimgeour, C.M. and Vedanayagam, H.S. (1977) Fatty acids. Part 50. 13C nuclear magnetic resonance studies of olefinic fatty acids and esters. Chem. Phys. Lipids, 18, 115129.
  • Gunstone, F.D., Steward, S.R., Cornelius, J.A. and Hammonds, T.W. (1972) New tropical seed oils. Part IV. Component acids of leguminous and other seed oils including useful sources of crepenynic and dehydrocrepenynic acid. J. Sci. Food Agric. 23, 5360.
  • Imoto, S. and Ohta, Y. (1988) Elicitation of diacetylenic compounds in suspension cultured cells of eggplant. Plant Physiol. 86, 176181.
  • Jacobs, J.J., Arroo, R.R., De Koning, E.A., Klunder, A.J., Croes, A.F. and Wullems, G.J. (1995) Isolation and characterization of mutants of thiophene synthesis in Tagetes erecta. Plant Physiol. 107, 807814.
  • Jung, R. and Kinney, A.J. (2001) Hypoallergenic transgenic soybeans. World Patent Application PCT WO 01/68887.
  • Kemp, M. (1978) Falcarindiol: an antifungal polyacetylene from Aegopodium podagraria. Phytochemistry, 17, 1002.
  • Kirsch, C., Hahlbrock, K. and Somssich, I.E. (1997a) Rapid and transient induction of a parsley microsomal Δ12 fatty acid desaturase mRNA by fungal elicitor. Plant Physiol. 115, 283289.
  • Kirsch, C., Takamiya-Wik, M., Reinold, S., Hahlbrock, K. and Somssich, I.E. (1997b) Rapid, transient, and highly localized induction of plastidial ω-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum. Proc. Natl. Acad. Sci. USA, 94, 20792084.
  • Kirsch, C., Takamiya-Wik, M., Schmelzer, E., Hahlbrock, K. and Somssich, I.E. (2000) A novel regulatory element involved in rapid activation of parsley ELI7 gene family members by fungal elicitor or pathogen infection. Mol. Plant Path. 1, 243251.
  • Lee, M., Lenman, M., Banas, A. et al. (1998) Identification of non-heme di-iron proteins that catalyze triple bond and epoxy group formation. Science, 280, 915918.
  • Van De Loo, F.J., Broun, P., Turner, S. and Somerville, C. (1995) An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc. Natl. Acad. Sci. USA, 92, 67436747.
  • Mancl, M.K. and Shein, S.E. (1982) Field Inoculation of Sunflower for Sclerotinia sclerotiorum Basal Stalk Rot and Virulence of Isolates From Various Hosts. Proceedings of the 10th International Sunflower Conference, Surfers Paradise, Australia, pp. 167169.
  • Mikolajczak, K.L., Smith, C.R., Jr, Bagby, M.O. and Wolff, I.A. (1964) A new type of naturally occurring polyunsaturated fatty acid. J. Org. Chem. 29, 318322.
  • Muir, A.D. and Majak, W. (1983) Allelopathic potential of diffuse knapweed (Centaurea diffusa) extracts. Can. J. Plant Sci. 63, 989996.
  • Nakano, Y., Matsunaga, H., Saita, T., Mori, M., Katano, M. and Okabe, H. (1998) Antiproliferative constituents in Umbelliferae plants. Part II. Screening for polyacetylenes in some Umbelliferae plants, and isolation of panaxynol and falcarindiol from the root of Heracleum moellendorffii. Biol. Pharmaceut. Bull. 21, 257261.
  • Nitz, S., Spraul, M.H. and Drawert, F. (1990) C17 polyacetylenic alcohols as the major constituents in roots of Petroselinum crispum Mill. Ssp. Tuberosum. J. Agric. Food Chem. 38, 14451447.
  • Olsson, K. and Svensson, R.S. (1996) The influence of polyacetylenes on the susceptibility of carrots to storage diseases. J. Phytopath. 144, 441447.
  • Pang, Z. and Sterner, O. (1991) Cibaric acid, a new fatty acid derivative formed enzymatically in damaged fruit bodies of Cantharellus cibarius (Chanterelle). J. Org. Chem. 56, 12331235.
  • Qiu, X., Reed, D.W., Hong, H., MacKenzie, S.L. and Covello, P.S. (2001) Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase. Plant Physiol. 125, 847855.
  • Reymond, P. and Farmer, E.E. (1988) Jasmonate and salicylate as global signals for defense gene expression. Curr. Opin. Plant Biol. 1, 404411.
  • Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406425.
  • Shanklin, J. and Cahoon, E.B. (1998) Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 49, 611641.
  • Shure, M., Wessler, S. and Federoff, N. (1983) Molecular characterization of the Waxy locus in maize. Cell, 35, 225233.
  • Somssich, I.E., Bollman, J., Hahlbrock, K., Kombrink, E. and Schulz, W. (1989) Differential early activation of defense-related genes in elicitor-treated parsley cells. Plant Mol. Biol. Rep. 12, 227234.
  • Towers, G.H.N., Page, J.E. and Hudson, J.B. (1997) Light-mediated biological activities of natural products from plants and fungi. Curr. Org. Chem. 1, 395414.
  • Villegas, M., Vargas, D., Msonthi, J.D., Marston, A. and Hostettmann, K. (1988) Isolation of the antifungal compounds falcarindiol and sarisan from Heteromorpha trifolata. Planta Med. 54, 3637.
  • Vlahov, G. (1996) Fatty acid distribution in triacylglycerols from aril and cotyledon oils of Afzelia cuanzensis. Phytochemistry, 42, 621625.
  • Voelker, T. and Kinney, A.J. (2001) Variations in the biosynthesis of seed-storage lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 335361.
  • DeWit, P.J.G.M. and Kodde, E. (1981) Induction of polyacetylenic phytoalexins in Lycopersicon esculentum after inoculation with Cladosporium fulvum (syn Fulvia fulva). Physiol. Plant Pathol. 18, 143148.
  • Zhu, L. and Minto, R.E. (2001) Improved syntheses of methyl (14E)- and (14Z)-dehydrocrepenynate: key intermediates in plant and fungal polyacetylene biosynthesis. Tetrahedron Lett. 42, 38033805.

GenBank accession numbers: AY166772, AY166773, AY166776, AY166778, AY16777, AY166774, and AY166775.