THE JOURNAL OF BIOLOGICAL CHEMISTRY 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 1, Issue of January 4, pp. 416 423, 2002 Printed in U.S.A.

Lipoxygenase H1 Gene Silencing Reveals a Specific Role in Supplying Fatty Acid Hydroperoxides for Aliphatic Aldehyde Production*
Received for publication, August 13, 2001, and in revised form, October 22, 2001 Published, JBC Papers in Press, October 23, 2001, DOI 10.1074/jbc.M107763200

Jose Leon, Joaqun Royo, Guy Vancanneyt , Carlos Sanz**, Helena Silkowski, Gareth Griffiths, and Jose J. Sanchez-Serrano
From the Centro Nacional de Biotecnologa, Consejo Superior de Investigaciones Cientficas, Campus de Cantoblanco, Universidad Autonoma de Madrid Colmenar Viejo km 15,500, 28049 Madrid, Spain, the **Instituto de la Grasa, Consejo Superior de Investigaciones Cientficas, Avenida Padre Garca Tejero 4, 41012 Sevilla, Spain, and Horticulture Research International, Wellesbourne, Warwick 35CV 9EF, United Kingdom

Lipoxygenases catalyze the formation of fatty acid hydroperoxide precursors of an array of compounds involved in the regulation of plant development and responses to stress. To elucidate the function of the potato 13-lipoxygenase H1 (LOX H1), we have generated transgenic potato plants with reduced expression of the LOX H1 gene as a consequence of co-suppression-mediated gene silencing. Three independent LOX H1-silenced transgenic lines were obtained, having less than 1% of the LOX H1 protein present in wild-type plants. This depletion of LOX H1 has no effect on the basal or wound-induced levels of jasmonates derived from 13-hydroperoxylinolenic acid. However, LOX H1 depletion results in a marked reduction in the production of volatile aliphatic C6 aldehydes. These compounds are involved in plant defense responses, acting as either signaling molecules for wound-induced gene expression or as antimicrobial substances. LOX H1 protein was localized to the chloroplast and the protein, expressed in Escherichia coli, showed activity toward unesterified linoleic and linolenic acids and plastidic phosphatidylglycerol. The results demonstrate that LOX H1 is a specific isoform involved in the generation of volatile defense and signaling compounds through the HPL branch of the octadecanoid pathway.

Lipoxygenase (LOX)1 enzymes catalyze the stereospecific dioxygenation of unsaturated fatty acids with a 1,4-pentadiene
* This work was supported by Spanish Comision Interministerial de Ciencia y Tecnologa Grant BIO99-1225, by Spanish Ministerio de Educacion y Ciencia postdoctoral contracts (to J. L. and J. R.) and postdoctoral fellowship (to G. V.), and by the Spanish Ministerio de Educacion y Ciencia-British Council Acciones Integradas Program (to J. J. S.-S. and G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Inst. de Biologia Molecular y Celular de Plantas, Universidad Politecnica de Valencia, Consejo Superior de Investigacio nes Cientficas, 46022 Valencia, Spain. Present address: Dept. de Biologa Celular y Genetica, Universidad de Alcala de Henares, 28871 Madrid, Spain. Present address: Aventis CropScience, B-9000 Ghent, Belgium. To whom correspondence should be addressed. Tel.: 34-915854500; Fax: 34-91-5854506; E-mail: jjss@cnb.uam.es. 1 The abbreviations used are: LOX, lipoxygenase; JA, jasmonic acid; HPL, hydroperoxide lyase; GC, gas chromatography; MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; TLC, thin layer chromatography; PIN2, proteinase inhibitor II; gfw, gram(s) fresh weight; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; TBS, Tris-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

system. C18 unsaturated fatty acids, linoleic acid (18:2 9,12) and linolenic acid (18:3 9,12,15), are major LOX substrates in plants. The lipoxygenase pathway of fatty acid metabolism (1) is initiated by the addition of molecular oxygen at the C9 or C13 position of the acyl chain yielding the corresponding 9- and 13-hydroperoxides (2). Both 9- and 13-hydroperoxides can subsequently be cleaved to short-chain oxoacids and aldehydes by the action of hydroperoxide lyases (HPL) or, alternatively, the 13-hydroperoxide is converted, after enzymatic cyclization, reduction, and -oxidation, to JA (3). Plant LOXs are ubiquitous and encoded by multigene families (4). The presence in a given tissue of several LOX isoforms with different substrate preferences, kinetic parameters, stereospecificity in substrate oxygenation, pH dependence, and subcellular localization makes difficult the assignment of specific functions to each LOX isoform. Moreover, LOX expression in plants is regulated throughout development and in response to stress (5, 6). Different LOX isoforms may have different physiological roles; they may, on the one hand, be responsible for the production of signals involved in the regulation of plant growth and the activation of stress-induced defense responses, whereas, on the other, the products of LOX activity may exert a direct deterring function toward pests and/or pathogens. It has been proposed that some aldehydes, in particular hexanal and hexenals produced by the action of HPL on LOX-derived fatty acid hydroperoxides, may be involved in the interaction between plants and pathogens or parasites (710) and, more recently, in the regulation of wound-induced gene expression (11). JA is well established as a regulator of defense mechanisms against wounding, caused by mechanical damage, chewing insects, and pathogen attack (3, 1217). Three LOX gene families have been characterized in potato; LOX1 is mainly expressed in tubers and roots, whereas LOX2 and LOX3 are expressed in leaves and are wound- and JAinducible (18, 19). Several LOX1-derived cDNAs have been isolated and shown to code for proteins with 9-LOX activity. LOX2 and LOX3 families are represented each by a single cDNA, LOX H1 and LOX H3, respectively, encoding proteins with 13-LOX activity (19). LOX genes with a high degree of sequence similarity to potato LOX H1 and H3 have been identified in tomato (20). The expression of the LOX H3 homologue (TomLoxD) was induced in the leaves of tomato plants by wounding and methyl jasmonate, following time courses similar to its potato counterpart. The expression of the tomato LOX H1 homologue (TomLoxC) was shown to be constitutive in fruits but, in contrast to potato, it was not detected in either wounded or nondamaged leaves.
This paper is available on line at http://www.jbc.org

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LOX H1-depleted Transgenic Potato Plants

Both antisense and co-suppression-mediated depletion of LOX genes have proved useful tools to elucidate the function of specific LOX isoforms and their corresponding products in defense signaling. In Arabidopsis, co-suppression-mediated depletion of a specific LOX isoform led to a reduction in the wound-induced accumulation of JA (21). On the other hand, antisense-mediated depletion of LOX gene expression has successfully been used to establish the involvement of a LOX isoform in the incompatibility trait of a tobacco variety resistant to the fungus Phytophthora parasitica (22), and the instrumental role of LOX H3 in the regulation of wound-induced gene expression and susceptibility to insect attack in potato (23). Here we report a transgenic approach to elucidate the functional role of LOX H1 in growth and development of potato plants, and to assess its potential role in the response to mechanical damage. We have generated transgenic lines expressing the complete LOX H1 cDNA under the control of the cauliflower mosaic virus 35 S promoter. Three transgenic lines that have undergone silencing of the transgene expression and co-suppression of the endogenous LOX H1 gene, have been characterized in terms of plant development and wound-induced changes in gene expression patterns.
EXPERIMENTAL PROCEDURES

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Plant Material and TransformationPotato plants (Solanum tuberosum cv. Desiree) were grown in soil either in the greenhouse or in growth chambers at 22 C under a 16-h light/8-h darkness photoperiod. Plants were transformed as described (24) by cocultivation with Agrobacterium tumefaciens harboring, in the BIN19 vector (25), the complete LOX H1 cDNA (19) in sense orientation under the control of the 35 S cauliflower mosaic virus promoter and the 3 terminator sequence of the octopine synthase gene. Plant transformants were selected for resistance to 50 mg/liter kanamycin (Sigma) and, after rooting, transferred to soil and grown as described above. Selected transformed lines were propagated vegetatively by either tuber sowing or explant cuttings. When indicated, potato leaves were wounded as described previously (19), and plant material harvested at the indicated times after wounding. RNA and Protein AnalysisTotal RNA isolation and Northern blot techniques were performed as described previously (19). Quantitation of PIN2 and LOX H3 RNAs was done by densitometry of autoradiograms derived of four independent experiments, using the Molecular Analyst program (Bio-Rad). Protein was extracted in 0.1 M Tris-HCl buffer, pH 7.2, containing 20% (v/v) glycerol, and electrophoretically separated in 10% PAGE gels under denaturing conditions (26). Separated proteins were then transferred to ECL-nitrocellulose membranes (Amersham Biosciences, Inc.), and subsequently hybridized with rabbit antibodies raised against LOX H1 protein or a specific peptide of LOX H3 as described previously (23), or against proteinase inhibitor 2 (kindly donated by Prof. Clarence A. Ryan, Washington State University, Pullman, WA). A peroxidase-coupled goat anti-rabbit antibody was used to detect immunoreactive proteins by the enhanced chemiluminescence system (Amersham Biosciences, Inc.). Chloroplast IsolationTwenty grams of potato leaves were harvested and directly homogenized in a blender in 200 ml of ice-cold extraction buffer (0.35 M sorbitol, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.1% bovine serum albumin, 15 mM -mercaptoethanol). The homogenate was filtered through two layers of Miracloth (Calbiochem) and centrifuged (1000 g) for 10 min at 4 C. The pellet was gently resuspended with a paint brush in 5 ml of ice-cold suspension buffer (0.3 M sorbitol, 20 mM Tricine-KOH, pH 7.6, 5 mM Mg2Cl, 2.5 mM EDTA), and poured onto a Percoll (Amersham Biosciences, Inc.) gradient, prepared on the previous day as follows; 30 ml of 50% Percoll in suspension buffer were centrifuged 30 min at 43,000 g at 4 C and kept overnight at 4 C in the centrifuge tubes. Separation of a layer with intact chloroplasts was achieved after 10 min of centrifugation at 13,200 g at 4 C in a HB-6 rotor (Sorvall). The chloroplast layer was carefully withdrawn with a glass pipette, and the chloroplasts washed in 20 ml of ice-cold suspension buffer. After 10 min of centrifugation at 2000 g at 4 C, the chloroplasts in the pellet were lysed in 1 ml of TE buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA), and protein concentration was determined by the Bradford method (Bio-Rad). Immunohistochemical Localization of LOX H1 ProteinWounded

and nonwounded leaflets of potato leaves, from wild-type and LOX H1-co-suppressed transgenic plants, were fixed by vacuum infiltration of square pieces (5 mm/side) with a 2% (w/v) paraformaldehyde solution in 0.1 M Tris-HCl buffer, pH 7.2, and then thoroughly washed three times with water. After fixation, leaf samples were progressively dehydrated by incubating for 1 h at room temperature in 30, 50, 70, 85, and 100% (v/v) ethanol. Dehydrated tissue was treated twice with xylene for 1 h each and then embedded in molten warm paraffin overnight at 42 C. Paraffin blocks, containing tissue samples, were formed by decreasing temperature and subsequently used to cut strips of 8- m sections with a microtome (Leica). Sections were laid on 2% triethoxysilyl-propylamino-treated glass microscope slides for further processing. Before immunolocalization procedure, tissue sections were deparaffinized by immersion twice in xylene and then thoroughly washed with TBS. Sections were blocked by incubation in TBS containing 1% (w/v) bovine serum albumin and 0.1% (v/v) Tween 20 for 3 h at room temperature. After washing three times with TBS containing 0.1% (v/v) bovine serum albumin and 0.1% (v/v) Tween 20 (washing buffer), sections were incubated with a 1:1000 dilution of antibody against LOX H1 in washing buffer for 1.5 h. After three new rounds of washing as described above, tissue sections were incubated in a high humidity closed plastic chamber with 0.1 ml of a 1:40 dilution of a 5- m gold particle-coupled anti-rabbit antibody (Sigma) in washing buffer for 1 h. Gold-labeled proteins were then treated with a silver enhancing kit (Amersham Biosciences, Inc.) as described by the manufacturers. Labeled proteins were observed and photographed by using a Nikon microscope equipped with an IGS filter that allows detection of epipolarized fluorescence. Determination of Endogenous Volatiles and Jasmonates from Potato LeavesTwenty-four discs were cut with a 5-mm cork borer from different leaves of potato plants, and immediately stored in liquid N2. Endogenous volatiles were quantified in quadruplicate by placing 24 leaf discs in an 11-ml vial containing 2.1 ml of saturated CaCl2 solution. The vial was then transferred into an automatic headspace sampler (Hewlett-Packard 19395A), where a 20-min equilibrium time at 80 C was set to allow endogenous volatiles to enter the gas phase. The volatiles were determined by gas liquid chromatography in a gas chromatograph (Hewlett-Packard 5890-II) equipped with a flame ionization detector and a glass column (2 mm 1 m) containing 5% Carbowax 20 M on 60/80 Carbopack B as the stationary phase. Column temperature was held isothermally at 120 C, injector at 150 C, and detector at 250 C. Carrier gas (N2) flow rate was 35 ml/min. Quantitation was performed by conversion of peak areas into nanomoles produced by the 24 leaf discs in 30 min by means of calibration curves obtained for the different compounds. Jasmonate contents in nonwounded and damaged leaves were determined by competitive enzyme-linked immunosorbent assay as described (23). LOX H1 Production in E. coliLOX H1 synthesis in BL21 E. coli cells and assays for LOX activity were essentially performed as described (19) with the exception that 20% glycerol and 0.01 mM phenylmethylsulfonyl fluoride were included in the buffer. LOX activity was determined spectrophotometrically by monitoring the increase in A234 resulting from conjugated diene formation from exogenously supplied lipid substrates containing polyunsaturated fatty acids provided as unesterified compounds or esterified to complex lipids extracted from potato leaf as described below. Typical assays contained 210 l of bacterial lysate supernatant, fatty acid, or complex lipid substrate (10 50 nmol, in ethanol, 0.01% final concentration) in 100 mM sodium acetate buffer, pH 6.0, at 25 C. Lipid Analysis and Substrate PreparationExtractions were performed using chilled solvents and glassware at 4 C. Lipids were extracted from 2 g of fresh leaf material in chloroform/methanol/0.15 M acetic acid (10/20/7.7, v/v) in a pestle and mortar and then an additional 10 ml of chloroform and 10 ml of water were added to achieve phase separation. The lower chloroform phase containing the complex lipids was removed and reduced to dryness under nitrogen. The residue was resuspended in a small volume of chloroform and the complex lipids separated by TLC in a chloroform/methanol/acetic acid/water (170/30/ 20/7, v/v). For quantitation, the complex lipids were transmethylated in situ in the silica gel using 2.5% sulfuric acid in methanol and the resulting methyl esters extracted into hexane and analyzed by GC using heptadecanoic acid as internal standard (27). For the preparation of lipid substrates for studies on LOX H1, after TLC, the lipids were eluted from the silica gel using the following solvents: acetone (100%) for DGDG, chloroform/acetone (50/50, v/v) for MGDG, chloroform/methanol (80/20, v/v) for PE, and chloroform/methanol (50/50, v/v) for PG and PC (28). For larger scale production of PG, complex lipids were ex-

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LOX H1-depleted Transgenic Potato Plants

FIG. 1. Analysis of LOX H1-silenced transgenic potato plants. A, Northern analysis of independent transgenic lines. Total RNAs were isolated from nonwounded leaves of transgenic plants and a non transformed plant (wt) as a control, and hybridization to the 32P-labeled NotI fragment containing the complete LOX H1 coding sequence was performed to detect endogenous gene and transgene transcripts, having the same size. B, wound-induced expression of LOX H1 gene. LOX H1 transcript was analyzed at different times in hours after wounding (h.a.w.) by Northern techniques with total RNA isolated from either wounded leaflets (local) or the nonwounded leaflets (systemic) of wounded leaves of wild-type (wt) and LOX H1-co-suppressed lines (#9, #31, and #33). Ethidium bromide-stained ribosomal RNA (rRNA) is shown as loading control.

FIG. 2. Expression of wound-inducible genes in wild-type and LOX H1co-suppressed potato plants. A, total RNA was isolated from wounded leaflets of wild-type (WT) and LOX H1-co-suppressed plants (#9, #31, and #33) at the indicated times (in hours) after wounding, and the levels of the transcripts from different wound-inducible genes were analyzed by Northern blot techniques using 32 P-labeled probes corresponding to lipoxygenases (LOX H1 and LOX H3), and proteinase inhibitor II (PIN2) cDNAs. Equal RNA loading was verified by ethidium bromide staining of the ribosomal RNA (rRNA) in the gel. B, quantitation of LOX H3 and PIN2 transcript accumulation in leaves of wild type (empty squares or circles) and LOX H1-co-suppressed line 31 (full squares or circles) upon wounding. RNA samples were taken at different times (in hours) after wounding. Values represent -fold induction over the levels determined in the nonwounded leaves of the corresponding plants and are the mean (bars represent standard deviations) of values obtained in four independent Northern blot assays.

tracted from 10 g of tissue and fractionated using DEAE column chromatography (28). The authenticity of PG was verified by TLC purification and GC analysis confirming the presence of its characteristic fatty acid, 16:1 (trans-3). The lipids were dried under nitrogen and resuspended in ethanol and stored under an atmosphere of nitrogen at 20 C until required.
RESULTS

Silencing of LOX H1 Gene in Transgenic Potato Plants Previously we have cloned and characterized a systemically induced 13-lipoxygenase (LOX H1) from potato leaves that is transcriptionally up-regulated in both wounded and nondamaged tissues (19). To ascertain the functional role of LOX H1, overexpression of the LOX H1 gene in transgenic potato plants was undertaken using a full cDNA under the control of the cauliflower mosaic virus 35 S promoter. Forty-eight independent transgenic lines were generated, and their basal level of LOX H1 transcript was compared with that of wild-type plants. Three of these lines (lines 9, 31, and 33) had much reduced LOX

H1 transcript levels in nonwounded leaves (Fig. 1A), which was suggestive of silencing or co-suppression of both transgene and endogenous gene expression. We tested whether silencing of LOX H1 gene in co-suppressed plants also affected its woundinduced expression. Local and systemic induction of LOX H1 expression upon wounding was detected in wild-type plants but was almost completely suppressed in the three transgenic lines analyzed (Fig. 1B). We also tested whether LOX H1 silencing affects the expression of LOX H3, another wound-inducible 13-lipoxygenase from potato leaves (19, 23). Fig. 2A shows that LOX H3 expression is induced upon wounding in the LOX H1-co-suppressed lines. Although in these plants LOX H3 transcript levels were lower than in the wild-type ones, the level of induction of LOX H3 gene expression in wild-type and co-suppressed plants was similar because of its lower basal transcription in the latter (Fig. 2B). However, the wound-induced accumulation of proteinase inhibitor 2 (PIN2) transcript was reduced in all LOX

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FIG. 3. Western analysis of lipoxygenase (LOX H1 and LOX H3) and proteinase inhibitor 2 (PIN2) proteins in wounded potato leaves from wild-type and LOX H1-co-suppressed plants. A, proteins were isolated from leaves of wild-type (wt) and LOX H1-co-suppressed lines (#9, #31, and #33) at different times after wounding (in hours, h.a.w.), separated in 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies that cross-react specifically to LOX H1, LOX H3, and PIN2. B, dilutions, from 1/50 to 1/1000, of the protein preparation corresponding to wt 24 h of panel A were used to compare the levels of the LOX H1 protein with those present in the leaves of silenced transgenic plants 24 h after wounding. Detection of the residual protein was possible by using at least 15 times longer exposure than that used for Westerns shown in panel A.

H1-co-suppressed lines (Fig. 2A). A more detailed analysis of line 31, the one showing the lowest LOX H1 levels (see below), revealed that wound-induced PIN2 transcript accumulation is similar up to 6 h but significantly lower at long times after wounding (24 and 48 h; Fig. 2B). These data indicate that LOX H1 is involved in signaling the wound-dependent PIN2 activation. The severely reduced accumulation of LOX H1 transcript in the co-suppressed plants prompted us to evaluate to what extent gene silencing had also led to a reduced accumulation of the corresponding protein product. Fig. 3A shows that, despite the increase in LOX H1 transcript accumulation observed upon mechanical damage, LOX H1 protein levels were not up-regulated in wounded leaves and remained nearly constant within the time frame analyzed, probably reflecting a balance between increase in transcription rate and protein turnover. Alternatively, translation of LOX H1 transcripts accumulating upon wounding may be delayed or inefficient within the time frame analyzed. Transgenic lines 9, 31, and 33, which did not accumulate LOX H1 transcript upon wounding (Figs. 1B and 2), had undetectable levels of protein whereas strong expression was seen in extracts from wild type plants (Fig. 3A). Only after long exposure times were residual levels of a cross-reacting protein detected in extracts from wounded leaves of co-suppressed plants. Quantitation of these residual levels indicated that line 33 had some 50 times less LOX H1 protein than wild-type plants by 24 h after wounding (Fig. 3B). Silencing of LOX H1 gene expression was more efficient in lines 9 and 31. The levels of LOX H1 protein in the wounded leaves of these lines were at least 100 times lower than in wild-type plants (Fig. 3B). In agreement with the levels determined for their corresponding transcripts, the reduced levels of LOX H1 protein in co-suppressed plants did not affect the levels of LOX H3 protein and resulted in a slight reduction of PIN2 protein levels (Fig. 3A). Developmental Control of LOX H1 Gene Silencing in Potato PlantsLOX H1 gene silencing in leaves of the co-suppressed transgenic line 31 appears to be subject to developmental reg-

FIG. 4. Developmental control of LOX H1 depletion in LOX H1-co-suppressed potato plants. Proteins were extracted from green flower buds (3 mm in diameter, lane 1) and from different leaves of increasing age, from the apex (lane 2) to fully expanded adult leaves (lane 8) of wild-type (WT) plants and the LOX H1-co-suppressed line 31. Proteins were also extracted from wild-type and line 31 flowers. Lane 1, green buds; lane 2, green buds (6 mm in diameter) with emerging petals; lane 3, buds (8 mm in diameter) with colored petals; lane 4, adult, open flower; lane 5, senescing flower. Western-type assay using specific antibodies to LOX H1 was conducted to determine LOX H1 protein content. The autoradiogram shown for line 31 flowers required 8 10-fold longer exposure times.

ulation, as indicated by the analysis of LOX H1 protein contents in the foliage, from actively growing apical leaves to adult fully expanded ones. In contrast to wild-type plants in which, as shown in Fig. 4, LOX H1 is constitutively present at similar levels in leaves at different developmental stages, LOX H1 gene expression appears to be silenced in fully expanded leaves (Fig. 4, lanes 37, #31 leaf) but only partially suppressed in actively growing, developing leaves (Fig. 4, lanes 2 and 3, #31 leaf) of line 31. LOX H1 gene is also expressed in flowers from potato plants (19) and the levels of protein do not change significantly at different stages of flower development (Fig. 4). We observed that gene silencing also occurred in the flowers of co-suppressed plants and was maintained throughout flower development (only shown for line 31). Phenotypic Characterization of LOX H1-co-suppressed Potato PlantsThe pattern and levels of LOX H1 expression suggested that it may have a function in plant development. Indeed, lines 9 and 31 have shorter internodes than wild-type plants, and smaller leaves, and branched and bushy plant shoots in which the axillary buds sprouted often (data not shown). As this altered phenotype is present in two independent co-suppressed lines, it likely relates to LOX H1 depletion and not to the result of somatic variations arising during the transformation procedure. However, line 33 did not exhibit any of these differences, perhaps because of its higher content in LOX H1 relative to lines 9 and 31. No alteration was observed in the root system of LOX H1-co-suppressed plants (data not shown). We also analyzed the production of tubers in wild-type and co-suppressed plants. Plants either grown from tubers or from explants were able to tuberize, and the total yield and number of tubers per plant in wild-type and co-suppressed plants were not significantly different (data not shown).

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FIG. 6. LOX H1 content in chloroplasts. LOX H1 content in protein extracts from total leaf tissue (T) and isolated chloroplasts (C) from wild-type (WT) and LOX H1-co-suppressed plants (#31) was compared by Western blotting using specific antibodies to LOX H1 (right panel). A replica gel was stained with Coomassie Blue (left panel) to show protein content and complexity of the respective extracts. The most intensely stained band corresponds to the Rubisco large subunit, which localizes to the chloroplast stroma. Molecular sizes (in kilodaltons) are marked on the left. The position of LOX H1 protein is marked on the right.

FIG. 5. Immunolocalization of LOX H1 protein in potato leaves. Leaf sections were incubated with a 1:1000 dilution of antibody against LOX H1 (CJ) or a pre-immune serum (A and B) as primary antibody, and a 1:40 dilution of a 5- m gold particle-coupled anti-rabbit antibody as secondary antibody. Green spots correspond to brightness originated from silver-enhanced gold-labeled proteins visualized with an IGS filter that allows detection of epipolarized fluorescence. AD, nonwounded wild-type leaf sections. E, F, I, and J, wounded leaves (24 h after wounding) of wild-type plants. G and H, wounded leaves (24 h after wounding) of LOX H1-co-suppressed plants (line 31). Immunostaining was detected by illumination with epipolarized light (B, D, F, H, and J) or a combination of epipolarized light with standard white light (A, C, E, G, and I). Tissue was stained with aniline blue. Original magnifications, 40 (AH) and 100 (I and J).

Immunolocalization of LOX H1 in Potato LeavesAnalysis of the deduced amino acid sequence of LOX H1 protein prompted the suggestion that LOX H1 gene product should be localized inside chloroplasts (19). We have now addressed the localization of LOX H1 by immunohistological techniques. The results shown in Fig. 5 indicate that, indeed, LOX H1 protein specifically accumulates in chloroplasts both in nonwounded and in damaged potato leaves (Fig. 5, CF) and also that no LOX H1 protein was detected in leaves of the co-suppressed line 31 (Fig. 5, G and H). The specific detection of LOX H1 protein was confirmed using the pre-immune serum that gave no signal in the immunolocalization procedure (Fig. 5, A and B). No LOX H1 protein outside the chloroplasts was detected in palisade parenchyma cells nor was it detected in epidermal cells (shown in the 100-fold magnification of Fig. 5, I and J).

Consistent with the data obtained in Western-type experiments (Fig. 3), no major wound-induced changes in LOX H1 protein amount were observed (compare panels D and F in Fig. 5). Chloroplastic localization of LOX H1 was confirmed by isolating intact chloroplasts from potato leaves and Western blot techniques (Fig. 6). As expected, chloroplasts isolated from the LOX H1-co-suppresed line 31 had a much reduced content of LOX H1 protein. Substrate Specificity of Recombinant LOX H1 ProteinIt is generally assumed that the substrates for LOX are unesterified polyunsaturated fatty acids released from complex lipids by the action of lipases and recently a role for phospholipase A2 acting on PC, analogous to mammals, has been proposed in plants (29, 30). However, LOX activity toward complex lipids has also been reported (4). In potato leaves the major complex lipids present are the galactolipids, MGDG (58.6 1.5%) and DGDG (24.8 1.4%), with the three main phospholipids being PC (7.6 1.1%), PE (4.4 0.6%), and PG (4.4 0.9%). PC and PE are predominantly found in the endoplasmic reticulum, whereas PG is the only phospholipid exclusively synthesized and located within the chloroplast (31). All complex lipids contained significant levels of 18:3, the fatty acid precursor of JA although 16:3, the precursor of dinor-JA (32) was a major acyl constituent in only MGDG and PG (Table I). These major complex lipids were purified from potato leaves in sufficient quantity necessary for substrate specificity studies of the cloned LOX H1. LOX H1 activity in extracts from transformed bacterial strains was highest toward the unesterified fatty acid substrates, linoleic acid and linolenic acid, which were utilized at similar rates (46.7 and 52.9 nmol of hydroperoxylinolenic acid formed/min/mg, respectively). When complex lipids were offered as substrates, no activity was detected with MGDG, DGDG, or PE. However, significant rates of activity were observed with PG, and in three independent preparations the rate was 16.7 9.5% of that observed for 18:3. Some activity toward PC was also observed but was 10 20% of that seen with PG as substrate. The activity of a commercially available lipoxygenase from soybean (Sigma) on PG as a substrate was also examined. Although good rates of activity were evident

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TABLE I Fatty acid composition of the major complex lipids in wild type potato leaves Lipids were extracted from freshly harvested leaf material in an acidified chloroform/methanol-based solvent (27). Lipids were purified by TLC and quantified as their fatty acid methyl esters by GC using heptadecanoic acid as internal standard. Values are presented as the mean of three independent samples standard error. Tr, trace amounts ( 0.5%). ND, not detected.
Fatty acid composition (mol %) Lipid 16:0 16:1 16:2 16:3 mol % 18:0 18:1 18:2 18:3

MGDG DGDG PG PC PE

1.9 7.8 15.1 21.2 22.8

0.4 0.9 1.5 2.0 1.2

Tr Tr 19.5 2.6 ND ND

Tr Tr Tr ND ND

31.7 2.7 2.9 0.5 27.1 3.1 ND ND

Tr 1.5 Tr 2.3 0.9 0.9 0.3 0.5 0.5 1.1 5.2 0.7

Tr 0.2 0.4 1.4 0.3

2.4 2.0 13.6 45.0 51.7

0.6 0.9 1.5 3.0 2.3

63.3 84.8 23.3 26.0 23.4

2.9 2.5 1.7 2.8 2.7

with unesterified fatty acids, no activity toward any complex lipid, including PG, could be demonstrated (data not shown). The Role of Fatty Acid Hydroperoxides Produced by LOX H1Lipoxygenases produce fatty acid hydroperoxides precursors for an array of different compounds (33). Biosynthesis of JA requires a 13-lipoxygenase activity (3). As LOX H1 catalyzes this type of reaction (19), we assessed the effect of LOX H1 depletion on basal and wound-induced JA levels in potato leaves. Jasmonate contents in the leaves of LOX H1-co-suppressed potato plants, as determined by competitive immunoassay (23), were not significantly different from those in wildtype plants both prior to and after wounding. A jasmonate content of 1.23 0.9 nmol/gfw was determined in nondamaged leaves of wild-type plants, and this level rose to 3.05 0.8 nmol/gfw 6 h after wounding. In the LOX H1-co-suppressed lines 9 and 31, the corresponding values in nondamaged leaves were 0.75 0.4 and 0.48 0.1 nmol/gfw, respectively, and 2.96 0.2 and 5.29 2.1 nmol/gfw 6 h after wounding. Although these results do not exclude its participation in jasmonate metabolism, they suggest that LOX H1 is not involved in the bulk production of jasmonates upon wounding. In addition to serving as precursors for jasmonate synthesis, 13-hydroperoxides of both linoleic and linolenic acids can be cleaved by HPL, yielding the C-6 aldehydes, hexanal and Z,3hexenal. We have determined the levels of these aldehydes, and the alcohols derived from them, in the leaves of wild-type and LOX H1-co-suppressed plants (lines 31 and 33). The results, summarized in Table II, show that plants with silenced expression of LOX H1 have less than 7 and 2% of hexanal and E,2-hexenal (the more stable isomer of Z,3-hexenal), respectively, of the levels determined for wild-type plants. Z,3-hexenol, which is the product of further reduction of E,2-hexenal by alcohol dehydrogenase, is present in lines 31 and 33 at less than 5% of the wild-type level, confirming that LOX H1-silenced plants are compromised in the production of C6-aldehydes and alcohols from the heterolytic cleavage of 13-hydroperoxides of linoleic and linolenic acids. In contrast, hexanal and E,2-hexenal levels in plants depleted for a different woundresponsive 13-LOX (LOX H3; line H3 4; Ref. 23) were similar to those found in wild-type plants. We have also determined the levels of C5-aldehydes and -alcohols, 1-penten-3-ol and Z,2pentenol, which are further oxidized to ethyl vinyl ketone and Z,2-hexenal, derived from lipoxygenase-mediated cleavage of 13-hydroperoxides of linoleic and linolenic acids (34). Those intermediates are present in H3 4 plants at around 80% of the levels detected in wild type plants, but all of them were significantly reduced in LOX H1-silenced plants. These data indicate that, in contrast to LOX H3, LOX H1 is an essential enzyme for the generation of C6- and C5-aldehydes and alcohols.
DISCUSSION

Fatty acid hydroperoxides and their metabolic derivatives (35, 36) are proposed to participate in the plant defense re-

sponse either as signaling compounds for activation of defense genes or as deterrents of insect attack and pathogen proliferation. In potato leaves, 13-hydroperoxides are generated by at least two different 13-LOX activities encoded in gene families (LOX2 and LOX3) with distinct patterns of induced expression upon mechanical damage. Antisense-mediated suppression of one of them (LOX3) showed that a specific LOX isoform (LOX H3) is required for mounting an efficient defense against insect herbivores, through induction of proteinase inhibitors and other defense-related genes (23). In this work, co-suppression of LOX2 gene expression, encoding a second 13-LOX isoform (LOX H1), reveals that the most likely role of LOX H1 is to supply HPL with the 13-hydroperoxy fatty acid substrates for the production of C-6 aldehydes, such as hexanal and 3-hexenal, and C-12 oxoacids. It has recently been shown that aphids grown in transgenic potato plants in which HPL has been depleted exhibit a much better performance than those maintained in wild-type plants (37). Because both LOX H1 and HPL are present in the leaves of healthy plants; this pathway is thus a constitutively deployed defense response toward sucking insects. Suppression of gene expression in transgenic plants stands as the method of choice for ascribing specific functions to gene products displaying similar activities. LOX H1-co-suppressed plants have provided a good model for the elucidation of its functional role because of the high selectivity of the co-suppression effect. Indeed, although LOX H1-co-suppressed plants have less than 1% of the protein detected in wild-type plants, they have standard levels of LOX H3 that is also induced in leaves in response to wounding (19). The LOX H1-depleted potato plants were obtained in an experiment designed to overexpress LOX H1 driven by the strong 35 S promoter from the cauliflower mosaic virus. Surprisingly, in none of the transformed lines could any significantly higher level of LOX H1 transcript accumulation be detected. In fact, three of them showed a drastic decrease in the level of LOX H1 transcript and protein, suggesting that transgene-mediated co-suppression of LOX H1 gene expression occurred. These observations suggest that potato plants may not tolerate high LOX H1 levels, which could compromise the viability of the plant by so far unknown mechanisms. As silencing of LOX H1 gene expression is observed in three independent transgenic lines, it can confidently be ascribed to the action of the introduced transgene. Co-suppression of LOX H1 leads to depletion of the protein in leaves and flowers of potato plants, but, remarkably, silenced expression was less evident in young actively growing apical leaves. Developmental regulation of silencing has already been reported (38). Although we cannot rule out the possibility that the developmental effects on LOX H1 co-suppression may actually be mediated by changes in the 35 S promoter activity (39, 40), limited co-suppression in actively growing leaves may in-

422

LOX H1-depleted Transgenic Potato Plants

TABLE II Content of C6- and C5-aldehydes and alcohols derived from cleavage of 13-hydroperoxides of linoleic and linolenic acids in wild type and LOX-silenced transgenic plants Nonwounded leaves of wild-type potatoes, and transgenic, LOX H3-silenced (H3 4; Ref. 23) and LOX H1 co-suppressed (lines 31 and 33) plants were used. Values presented are expressed in nmol/cm2 of leaf area, and represent the mean of five independent samples containing 24 leaf discs (5 mm in diameter) each standard error.
Wild-type H34 Line 31 Line 33

Z,3-Hexenol E,2-Hexenol Hexanal E,2-Hexenal Z,2-Pentenol Z,2-Pentenal 1-penten-3-ol Ethyl vinyl ketone

0.53 0.046 0.48 13.59 0.41 0.28 0.31

0.08 0.023 0.11 2.40 0.07 0.06 0.03

0.32 0.020 0.35 11.64 0.33 0.22 0.26

0.07 0.005 0.09 1.39 0.05 0.03 0.04

0.02 0.011 0.03 0.16 0.06 0.06 0.07

0.01 0.005 0.01 0.08 0.01 0.01 0.03

0.01 0.007 0.03 0.20 0.05 0.04 0.15

0.00 0.002 0.01 0.10 0.01 0.01 0.03

deed result from an essential role of LOX H1 at this developmental stage that requires (and tolerates) higher levels of the protein. In support of a function for LOX H1 in regulating growth and development, we have found that the aerial parts of some of the LOX H1-co-suppressed lines display a characteristic phenotype clearly distinguishable from that of wild-type plants. LOX H1co-suppressed shoots are smaller and more branchy than wildtype ones. A high number of actively growing axillary buds present in the co-suppressed shoots suggests that apical dominance is reduced in LOX H1-co-suppressed lines. This phenotypic alteration may be because of a direct effect of depleting any LOX H1-derived products or, alternatively, to a secondary effect mediated by alterations in the hormonal imbalance in the co-suppressed plants, as this phenotype resembles that of mutants or transgenic plants affected also in the balance of cytokinins, auxins, and/or brassinosteroids (Refs. 41 43; for a recent review on apical dominance and control of axillary bud growth, see Ref. 44). However, because one of the LOX H1depleted lines (line 33) does not show these phenotypic traits, it is unclear to what extent they may be directly related to the lower LOX H1 levels present in the other transgenic lines (lines 9 and 31). LOX H1 constitutively accumulates throughout flower development, and a role in supplying fatty acid hydroperoxide precursors for the production of volatiles to attract pollinators could be envisaged. LOX H1-co-suppressed plants tend to flower earlier than wild-type ones (data not shown). Remarkably, the early flowering Arabidopsis mutant efs has also a reduction in apical dominance (45), supporting a possible link between these processes and suggesting that a LOX H1-derived product may participate in their regulation. The presence of putative transit peptides in their deduced protein sequences suggested that LOX H1 resides in plastids (19). Indeed, chloroplast protein extracts are enriched in LOX H1 and, moreover, immunolocalization confirms the presence of LOX H1 exclusively in the chloroplasts. Immunohistology with specific antibodies also reveals that LOX H3 preferentially accumulates in chloroplasts (data not shown). In this regard, both potato 13-LOX have a similar subcellular localization to their corresponding homologues in tomato (20). Chloroplast localization is an important feature regarding the possible role of 13-LOX in the octadecanoid pathway (1). It has been proposed that chloroplasts are the major site for fatty acid hydroperoxide metabolism (46). Other wound and/or jasmonate-inducible lipoxygenases such as those of barley leaves are, as well, localized in the chloroplasts (47) or, in the case of the Arabidopsis AtLOX2, have typical chloroplast transit peptides (6). Because of its 13-LOX stereospecific activity using linoleic and linolenic acids as substrates, in addition to the transcriptional activation detected both in wounded and JAtreated potato leaves (19), both LOX H1 and H3 were good candidates to be involved in jasmonate synthesis in vivo. We

reported previously (23) that, despite its prominent role in plant resistance to pest attack, LOX H3 was not a rate-limiting activity in the wound-induced synthesis of JA. Our results indicate that wound-induced accumulation of jasmonates is not reduced in LOX H1-co-suppressed plants either, suggesting that LOX H1 is not implicated in the synthesis of the bulk of jasmonates in response to wounding. Instead of serving as JA precursors, LOX H1-derived fatty acid hydroperoxides are substrates for HPL and, thus, LOX H1-co-suppressed plants have much reduced levels of the oxylipins produced through this branch of the octadecanoid pathway, namely C6 aldehydes and alcohols and the corresponding oxoacid, traumatic acid. In contrast, C-6 aldehyde levels in LOX H3-depleted plants are nearly identical to wild-type. As both LOX H1 and H3 localize to chloroplasts, the hydroperoxide pool generated by LOX H1 should be compartmentalized for its exclusive use through the HPL catabolic pathway, whereas JA synthesis would depend on the hydroperoxide products of LOX H3 or another, hitherto uncharacterized, LOX activity. This compartmentalization could either depend on specific protein-protein interactions to generate metabolic chains or be because of a differential suborganellar distribution of the enzymes involved, thus restricting access to one anothers hydroperoxide pools. Both possibilities are currently being explored. It is generally assumed that the substrate for LOX is unesterified polyunsaturated fatty acids released from complex lipids by the action of lipases. However, LOX activity toward complex lipids has also been reported (4). Although unesterified fatty acids are the preferred substrate for the LOX H1 enzyme in vitro, it also shows significant activity on PG, the only phospholipid entirely synthesized within plastids (31). Because LOX H1 is targeted to the chloroplast, we considered that the galactolipids, which are the major thylakoid complex lipids and which are rich in 18:3, could be a suitable substrate. However, no activity toward these lipids was detected, suggesting that this LOX isoform does not act on the major membrane components of the chloroplast. LOX H1 activity on PG would thus generate hydroperoxides that are esterified to complex lipids. To be further processed down the octadecanoid pathway, such ester-linked hydroperoxides would have to be released by the action of a lipase with a specificity toward oxygenated fatty acids. In plants such an activity has been observed in the remodeling of PC by phospholipase A2 in tissues that accumulate high levels of the hydroxy-fatty acid, ricinoleic acid, in their seed oils (48). The significance of this activity of LOX H1 remains, at present, unclear, although the ability of a LOX to utilize this substrate offers a potential means of compartmentation and restriction of substrate to a relatively minor plastid phospholipid. However, whether the enzyme uses unesterified polyunsaturated fatty acids or polyunsaturated fatty acids esterified in PG will be dependent on the availability of both substrates to the enzyme in vivo. Conconi et al. (29) estimated that unesterified fatty acids levels increased from 75 to 125

LOX H1-depleted Transgenic Potato Plants

g/g dry weight 1 h after wounding and accounted for less than 0.25% of total fatty acids present in tomato leaves. Concomitantly, an increase in lyso-PC was observed suggesting that the 18:3 liberated for subsequent JA synthesis may arise from PC. In LOX H1-co-suppressed plants, however, no effect of transgene expression on basal or wound-induced levels of JA was observed. However, the severe reduction in volatile production observed in the LOX H1-co-suppressed plants suggests that the pool of lipid hydroperoxides generated by LOX H1 serve as substrates for HPL. Recently, we have shown that the basal level of hydroperoxides in potato leaf was 334 75 nmol gfw and are esterified to complex lipids (27). Thus, the substrate specificities of LOX H1 reported here suggest that PG could provide the hydroperoxide fatty acid substrate for HPL lyase leading to aliphatic aldehyde production. Both Western blotting and immunohistological detection indicate that LOX H1 accumulates at fairly high levels in nonwounded potato leaves. However, 9-LOX appears to be the predominant specific activity in the leaves of healthy, nondamaged potato plants (49). LOX H1 may thus be present in an inactive form in those leaves, and be post-translationally activated upon stress, or in other situations. LOX H1 could thus be involved in a defense response to pests and pathogens, different from that related to herbivory and involving activation of genes, proteinase inhibitors in particular. Consistent with that hypothesis, the wound-induced activation of LOX H3 gene and other wound-responsive genes such as allene oxide synthase (AOS; data not shown) is not significantly affected in LOX H1-co-suppressed plants. However, wound induction of the proteinase inhibitor II (Pin2) gene is partly reduced, perhaps reflecting the requirement for its full activation of a component that is not present in LOX H1-co-suppressed plants. It has been reported that C6 aldehydes may induce a subset of defenserelated genes such as those encoding enzymes of the phenylpropanoid pathway (11). However, reducing C6 aldehyde content through HPL depletion does not result in reduced levels of Pin2 transcripts upon wounding (37). Thus, LOX H1-derived compounds other than C6 aldehydes, or LOX H1 itself, may additionally play a role in the wound-induced activation of a subset of responsive genes. The use of NADPH oxidase inhibitors has revealed that hydrogen peroxide is required for the induction of a subset of wound-responsive genes, Pin2 among them, but not for wound-induced activation of LOX H3 and AOS gene expression (50). Similar to NADPH oxidase inhibition, the wound-induced activation of both subsets of genes is affected differentially in the LOX H1-co-suppressed plants, suggesting that LOX H1 activity is involved in gene induction through the H2O2-dependent pathway. The use of LOX H1-co-suppressed potato plants provides further insights on the role of LOX H1 in the synthesis of volatiles that are detrimental to pests and pathogens, as a prerequisite for genetic manipulation of plants toward high levels of active LOX H1.
AcknowledgmentsWe gratefully acknowledge Tomas Cascon and Pilar Paredes for excellent technical assistance, and Ines Poveda and Angel Sanz for the photographic work. We thank Prof. C. A. Ryan for potato pin2 antibodies, M. Dolores Gomez and Luis Canas (IBMCP, Valencia, Spain) for the assistance in immunohistology techniques, and Carmen Castresana for comments and suggestions on the manuscript.
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