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Originally published In Press as doi:10.1074/jbc.M207234200 on September 25, 2002

J. Biol. Chem., Vol. 277, Issue 48, 46051-46058, November 29, 2002
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Identification of a Jasmonate-regulated Allene Oxide Synthase That Metabolizes 9-Hydroperoxides of Linoleic and Linolenic Acids*

Aya ItohDagger §, Anthony L. SchilmillerDagger §||, Bonnie C. McCaigDagger , and Gregg A. HoweDagger ||**

From the Dagger  Department of Energy Plant Research Laboratory and || Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1312

Received for publication, July 18, 2002, and in revised form, September 24, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to alpha -ketol, gamma -ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was approx 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxylipins comprise a group of biologically active compounds that are produced by oxidative metabolism of polyunsaturated fatty acids. Members of the eicosanoid family of lipid mediators have been studied extensively with respect to their biosynthesis from arachidonic acid and their function in diverse physiological processes in animal cells (1). In plants, which lack arachidonic acid, oxygenated derivatives of C18 fatty acids participate in the regulation of many defense-related and developmental processes. The biosynthesis of most phytooxylipins is initiated by lipoxygenase (LOX),1 which adds molecular oxygen to either the C-9 or C-13 position of linolenic or linoleic acid (2). The resulting hydroperoxides are further metabolized by several enzymes including three closely related members of the CYP74 family of cytochromes P-450: allene oxide synthase (AOS), hydroperoxide lyase (HPL), and divinyl ether synthase (DES). Indeed, much of the structural and functional diversity in oxylipin metabolism in plants can be accounted for by the activity of CYP74s that metabolize 9- and 13-hydroperoxides to a wide range of products (3). In contrast to typical P-450 monooxygenases, CYP74 P-450s do not require O2 and a NADPH-dependent P-450 reductase for activity. Rather, they use a hydroperoxide group both as the oxygen donor and as a source of reducing equivalents (4, 5). This unique catalytic feature is shared by thromboxane synthase and prostacyclin synthase, two P-450 enzymes involved in the synthesis of eicosanoids (6). A greater understanding of the biochemical and physiological function of this atypical class of P-450 enzymes promises to provide new insight into the evolution of fatty acid-based signaling pathways in diverse biological systems.

Interest in plant oxylipins has focused mainly on the AOS branch of the 13-LOX pathway that gives rise to the jasmonate family of compounds (collectively referred to as JAs) including JA, methyl-JA (MeJA), and their metabolic precursor 12-oxo-10,15-phytodienoic acid (12-OPDA) (Fig. 1). AOS catalyzes the transformation of 13-hydroperoxy linolenic acid (13-HPOT) to a short-lived allene oxide intermediate, 12,13-epoxyoctadecatrienoic acid (12,13-EOT) (7, 8). In the biosynthetic route to JA, allene oxide cyclase (AOC) converts 12,13-EOT to 12-OPDA (9). Reduction of 12-OPDA by OPDA reductase (OPR) and three cycles of beta -oxidation yields JA (10, 11). During in vitro reactions carried out in the absence of AOC, 12,13-EOT spontaneously hydrolyzes to alpha - and gamma -ketols, and can also undergo non-enzymatic cyclization to produce racemic 12-OPDA (4, 8). The physiological significance of alpha - and gamma -ketols, and the extent to which they are produced in vivo, is unclear. Genes encoding AOSs that metabolize 13-HPOT have been cloned from several plant species. Based on comparisons of predicted amino acid sequence, these proteins are classified within the CYP74A subfamily of P-450s (2, 3). Several AOSs have been shown to have a strong (approx 36:1) preference for the 13-hydroperoxide relative to the corresponding 9-isomer (e.g. Refs. 12 and 13). One exception to this is barley AOS, which despite its classification as a CYP74A and proposed role in JA biosynthesis can metabolize both 9- and 13-isomers in vitro (14).

Plants also utilize a second type of AOS for the metabolism of 9-hydroperoxides in non-photosynthetic tissues (Fig. 1). An AOS activity responsible for production of alpha - and gamma -ketol fatty acids from 9-hydroperoxy linoleic acid (9-HPOD) was first demonstrated in extracts from corn seed (15, 16). Subsequent studies showed that these compounds are formed by non-enzymatic hydrolysis of an unstable allene oxide (9,10-epoxyoctadecanoic acid; 9,10-EOD) that is generated by the action of AOS on 9-hydroperoxides (17, 18). More recently, the production of ketols by the sequential action of 9-LOX and AOS was demonstrated in cell-free extracts from tomato root (19), tulip bulb (20), and potato stolon (18). The latter study (18) also showed that metabolism of linoleic and linolenic acids via the 9-LOX/AOS pathway gives rise to 10-oxo-11-phytoenoic acid (10-OPEA) and 10-oxo-11,15-phytodienoic acid (10-OPDA), respectively, novel cyclopentenones that are structural isomers of 12-OPDA (Fig. 1).


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  		Fig. 1.   The allene oxide synthase route of oxylipin metabolism. The pathways shown are for metabolism of linolenic acid by the AOS branch of the 9-LOX (left) and 13-LOX (right) pathways. See text for details.

The presence of 9-AOS in plants raises the question of the physiological function of this branch of oxylipin metabolism and its relationship to AOSs that act in the 13-LOX pathway for JA biosynthesis. Here we describe the identification and functional characterization of a cDNA encoding a novel member of the CYP74 P-450 gene family in tomato. We demonstrate that the recombinant protein (designated LeAOS3) is an AOS by virtue of its ability to convert hydroperoxy fatty acids to ketol and cyclopentenone oxylipins. The phylogenetic classification of LeAOS3 within the CYP74 family, the substrate preference of the enzyme and the tissue-specific expression of LeAOS3 transcript lead us to propose a role for this P-450 in the production of 9-hydroperoxide-derived oxylipins. We also demonstrate that expression of LeAOS3 mRNA in roots is abolished in a mutant of tomato that is insensitive to JAs. The relevance of these findings to the possible physiological function of LeAOS3 is discussed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plant Materials and Growth Conditions-- Tomato (Lycopersicon esculentum cv. Castlemart) plants were grown in Jiffy peat pots (Hummert International) in a growth chamber maintained under 17 h of light (200 µE2/s) at 28 °C and 7 h of dark at 18 °C. Seeds of Lycopersicon pennellii (LA716) and the introgression lines used for restriction fragment length polymorphism (RFLP) mapping were obtained from the Tomato Genetics Resource Center (Davis, CA). Plants homozygous for jai1-1 were obtained from a population of F2 plants that was segregating the mutation, as previously described (21).

Isolation of a Full-length LeAOS3 cDNA-- Basic molecular biological techniques were performed as described in Sambrook et al. (22). The expressed sequence tag (EST) clone cLEI16I12 (GenBankTM AW651347; EST329801) was obtained from the Clemson University Genomics Institute. The cDNA insert, which was sequenced in its entirety on both strands, was 1773 base pairs (bp) in length, and included 16-bp upstream of the initiator AUG codon and 281 bp in the 3'-untranslated region (excluding poly(A) residues). Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was used to obtain additional sequence information at the 5'-end of the gene as previously described (23). 20 ng of tomato genomic DNA was used as a template for an initial PCR reaction using a gene-specific primer (GSP1: 5'-TGG-AGT-TGT-AGA-ACG-CGT-TGT-ATA-GCT-TC-3') and a shorter arbitrary degenerate primer (AD1: 5'-(A/C/G/T)TCGA(C/G)T(A/T)T(C/G)G(A/T)GTT-3'). The resulting PCR product was used as a template for a second PCR reaction using the same AD1 primer and a second nested gene-specific primer (GSP2: 5'-CTG-ATG-ATC-GCG-TTT-TAC-AAG-GAT-GAT-AG-3'). The PCR products obtained from this reaction were excised from the gel and used as template for a final PCR reaction with the AD1 primer and a third nested gene-specific primer (GSP3: 5'-CCT-AGT-GAA-ATC-AAT-GGA-GCT-AGT-TGA-AG-3'). TAIL-PCR products of ~850 bp were obtained from two independent reactions. DNA sequencing of these products was performed using a gene-specific primer (5'-GAT-GTA-GTA-AAG-ATG-GGA-TG-3'), and revealed an in-frame stop codon (UAA) 42 nucleotides upstream of the initiator AUG. Reverse transcription PCR was used to exclude the possibility that the in-frame stop codon was located in an intron that was amplified by TAIL-PCR. Reverse transcription was performed with oligo(dT) primers and Enhanced Avian reverse transcriptase (Sigma Chemical Co.). PCR was performed using a primer (5'-GTG-ATT-ATT-CTA-ATC-TCT-AGC-ACT-ATC-TA-3') located upstream of the in-frame stop codon and the GSP3 primer. Direct sequencing of the RT-PCR product confirmed the position of the stop codon relative to the initiator AUG codon. Data base searches were performed using the BLAST program (24) available at the United States National Center for Biotechnology. Amino acid sequence alignments were performed using the Clustal method in the Megalign program (DNAStar, Madison, WI). An unrooted neighbor-joining phylogeny was constructed in PAUP4.0* version 4.0b10 (25), and 100 bootstrap replicates were run to test the reliability of the tree topology.

Expression and Purification of Recombinant LeAOS3-- A PCR-based approach was used to construct a vector for expression of LeAOS3 containing the C-terminal His6 tag. Forward and reverse primers that amplify the cDNA (EST clone cLEI16I2) were designed to contain NdeI and XhoI restriction sites, respectively. The sequence of the forward primer was 5'-GGA-ATT-CCA-TAT-GGC-TAA-TAC-CAA-AGA-3' and that of reverse primer, 5'-CCG-CTC-GAG-TGA-TGT-TGC-TTT-AG-3'. PCR amplification yielded a 1.45-kb product that was subsequently cut with NdeI and XhoI and subcloned into the same sites of the expression vector pET23-b (Novagen, Madison, WI). The resulting construct, which added eight amino acids (LEHHHHHH) to the C terminus of LeAOS3, was transformed into E. coli host strain BL21(DE3).

His-tagged recombinant LeAOS3 was expressed in BL21(DE3) host cells as follows. An overnight culture (2 ml) of bacteria was inoculated into 200 ml of Terrific Broth (TB) medium supplemented with 100 µg/ml ampicillin. Bacteria were grown at 37 °C in a shaker at 250 rpm to an OD600 of 0.5. Cultures were cooled to 25 °C, and isopropyl-thio-beta -D-galactopyranoside was added to a final concentration of 0.1 mM. Induced cultures were incubated for 24 h at 25 °C with gentle shaking (120 rpm). Cells were collected by centrifugation and stored at -20 °C until further use. Purification of recombinant LeAOS3 was performed as described previously (23) with minor modification. Following centrifugation (100,000 × g for 60 min) of the cleared E. coli lysate, the recombinant protein was solubilized from the particulate fraction using 1.5% Triton X-100R. Solubilized protein was further purified using TALON metal affinity column (cobalt-based IMAC, Clontech) and subsequent elution with imidazole. Imidazole was removed from the protein sample using a 2.5-ml spin column prepared with Sephadex G-25 (Amersham Biosciences) equilibrated with 50 mM sodium phosphate (pH 7.0), 5% glycerol, and 0.02% Triton X-100R. Protein measurements were performed using a BCA assay (Pierce) and bovine serum albumin as a standard. The relative purity of recombinant LeAOS3 was estimated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) and staining of gels with Coomassie Brilliant Blue R-250.

Product Analyses-- Affinity-purified LeAOS3 (30 µg of protein) was incubated with 250 µg of fatty acid hydroperoxide substrate (Cayman Chemical, Ann Arbor, MI) in 1 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 5% (v/v) glycerol. Reactions proceeded for 5 min at 25 °C and then were stopped by acidification to pH 4.0 with 1 M citrate. Products were extracted twice with chloroform, dried under N2 gas, and resuspended in 0.25 ml of methanol. Compounds were methylated by treatment with ethereal diazomethane at 25 °C for 10 s and dried under N2 gas. Samples subjected to silylation were treated with 30 µl of the following mixture: pyridine/hexamethyldisilazane/trimethylchlorosilane at a ratio of 2:1:2 (v/v/v) at 25 °C for 20 min (26). Following removal of excess reagent under vacuum, the remaining residue was dissolved in hexane and analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described (13), with the following modifications. The temperature program was initiated at 50 °C, ramped to 320 °C at 10 °C min-1, and maintained at 320 °C for 2 min. Stereochemical analysis of products was not attempted. However, it was noted that incubation of LeAOS3 with hydroperoxides yielded two cyclopentenone products, presumably diastereomers, which were separable by GC and gave identical mass spectra. The peak area of these two compounds was added together to compute the total cyclopentenone composition. The mass spectrum of 12-OPDA produced from LeAOS3-catalyzed metabolism of 13-HPOT was identical to that of an authentic 12-OPDA standard (Cayman Chemical).

Biochemical Analysis of LeAOS3-- The hydroperoxide-metabolizing activity of recombinant LeAOS3 was measured spectrophotometrically by monitoring the rate of decrease in absorbance at 234 nm resulting from disruption of the conjugated diene bond of the substrate (27). Kinetic assays were performed at 30 °C in 1 ml of 100 mM sodium phosphate (pH 7.0) containing 30 ng or 60 ng of purified LeAOS3 and varying concentrations of hydroperoxide substrate. Activity slopes obtained during the first 0.5 min of the reaction were used for calculation of kinetic parameters. Absorbance spectra were obtained using purified recombinant protein in 1 ml of 100 mM sodium phosphate buffer (pH 7.0). Carbon monoxide treatments were performed by bubbling CO gas through the sample for 1 min. The protein was reduced by the addition of a few grains of sodium dithionite.

RNA Blot Analysis and Gene Mapping-- RNA and DNA blot analyses were performed as previously described (13, 23). Total RNA was extracted from soil-grown plants or from seeds (4 days after imbibition) that were germinated on water-saturated filter paper. Hybridization signals on RNA blots were normalized to the signal obtained using a cDNA probe for translation initiation factor eIF4A mRNA, obtained from Clemson University (EST clone cLED1D24). Chromosome mapping of LeAOS3 was performed as previously described (23), using a set of introgression lines that harbor defined segments of L. pennellii DNA in an otherwise L. esculentum genetic background (28). A RFLP that distinguishes LeAOS3 in L. pennellii from its homolog in L. esculentum was generated by digestion of genomic DNAs with DraI. LeAOS3 was mapped to the IL10-1 interval on chromosome 10 (Tomato Genetics Stock Center accession number LA3515). Hybridization of genomic DNA blots to an RFLP marker (TG103) known to map to this interval confirmed the identity of LA3515.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a New Member of the CYP74 Gene Family in Tomato-- A search of the tomato EST data base (www.tigr.org/tdb/lgi/) was conducted to identify potential new members of the CYP74 gene family in tomato. A tentative consensus sequence (29), constructed from multiple overlapping EST clones, was identified that was similar to but clearly distinct from previously characterized CYP74 sequences in tomato and other plant species. DNA sequence analysis of one EST clone (cLEI16I12) revealed a 1772-bp cDNA insert that contained an open reading frame predicted to encode a 491-amino acid protein, having a calculated molecular weight of 55,513. In keeping with the previous nomenclature of tomato CYP74s (3) and the biochemical characteristics of the protein (see below), we henceforth refer to the cDNA as LeAOS3 (L. esculentum AOS3). The deduced amino acid sequence of LeAOS3 showed several features that distinguish CYP74s from P-450 monooxygenases (Fig. 2). For example, the I-helix region of P-450 monooxygenases contains an invariant threonine residue that serves an important role in the binding and activation of oxygen (30, 31). All reported CYP74s, including LeAOS3, contain a small hydrophobic residue (I/A/V) at this position. The CYP74 consensus sequence for residues surrounding the cysteinyl heme ligand near the C terminus is NKQC(A/P)(G/A)K(D/N)XV. With the exception of a V right-arrow I substitution in the last position, this sequence is conserved in LeAOS3. Analysis of the deduced amino acid sequence of LeAOS3 using the ChloroP program (www.cbs.dtu.dk/services/ChloroP/) suggested that the protein does not contain a predicted N-terminal targeting sequence that directs many, but not all (32, 14), CYP74 P-450s to the chloroplast.


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  		Fig. 2.   Comparison of cDNA-deduced protein sequences of CYP74 P450s in tomato. Sequences were aligned using the ClustalW1.8 program available at SearchLauncher.bcm.tmc.edu. Sequences shown are LeAOS1 (AJ271093; CYP74A), LeAOS2 (AF230371; CYP74A), LeAOS3 (AF454634; CYP74C), LeHPL (AF230372; CYP74B), and LeDES (AF317515; CYP74D). Black and shaded boxes in the alignment indicate positions that contain an identical and conserved amino acid, respectively. The and  symbol denotes the position of the conserved threonine found in the I-helix of P-450 monooxygenases. The CYP74 consensus sequence surrounding the cysteinyl heme ligand (*) is underlined.

Phylogenetic analysis was used to gain insight into the functional relationship between LeAOS3 and extant members of the CYP74 family, which is comprised of four subfamilies (CYP74A through CYP74D). An unrooted neighbor-joining phylogeny indicated that CYP74 members fall into distinct groups that reflect, to a large extent, the known enzymatic identity and substrate specificity of individual members (Fig. 3). This phylogeny was in perfect agreement with that obtained using a maximum parsimony algorithm (not shown). The CYP74A subfamily, which forms a monophyletic cluster with monocots positioned basally to the dicots, consists mainly of AOSs that have specificity for 13-hydroperoxides (i.e. 13-AOSs). The CYP74B subfamily also forms a well defined group comprised of HPLs that have specificity for 13-hydroperoxides (i.e. 13-HPLs). CYP74C P450s from cucumber (CsHPL) and melon (CmHPL) are HPLs that have a preference, but not absolute specificity, for 9-hydroperoxides (33, 34). Members of the CYP74D subfamily are DESs that have high specificity for 9-hydroperoxides (23, 35). LeAOS3 was most similar to the mixed specificity HPLs (CYP74Cs) from melon (58% identity) and cucumber (57% identity). In accordance with cytochrome P-450 nomenclature, LeAOS3 was classified as a CYP74C and was assigned the name CYP74C3.2


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  		Fig. 3.   Phylogenetic tree of the CYP74 family of cytochromes P-450. An unrooted neighbor-joining phylogeny was constructed in PAUP4.0* from the deduced amino acid sequences of representative CYP74 P-450s. The groups enclosed by circles indicate the four CYP74 subfamilies (CYP74A through CYP74D). Numbers indicate percent bootstrap support for each branch of the phylogeny. AOS sequences were from barley (HvAOS1, AJ250864; HvAOS2, AJ251304), tomato (LeAOS1, AJ271093; LeAOS2, AF230371; LeAOS3, AF454634), Arabidopsis (AtAOS, Y12636), guayule (PaAOS, X78166), flax (LuAOS, U00428), rice (OsAOS, AY055775), tobacco (NaAOS, AJ295274) and potato (StAOS1, AJ457080; StAOS2, AJ457081). HPL sequences were from tomato (LeHPL, AF230372), bell pepper (CaHPL, U51674), Arabidopsis (AtHPL, AF087932), Medicago sativa (MsHPL, AJ249245), tobacco (NaHPL, AJ414400), potato (StHPL, AJ310520), melon (CmHPL, AF081955), and cucumber (CsHPL, AF229811). DES sequences were from tomato (LeDES, AF317515), potato (StDES, AJ309541), and tobacco (NtDES, AF070976).

Biochemical Properties of LeAOS3-- The full-length LeAOS3 cDNA was subcloned into an expression vector (pET23-b) that adds a His6 tag to the C terminus of the protein. Following introduction of this construct into E. coli, recombinant LeAOS3 was expressed and purified using metal affinity chromatography. The majority of LeAOS3 was found in inclusion bodies in the 3,000 × g pellet after centrifugation of sonicated cells. Approximately 80% of enzyme activity found in the 3,000 × g supernatant was subsequently recovered in the 100,000 × g pellet, indicating that the active protein is associated with bacterial membranes. Solubilization of membranes with Triton X-100R followed by cobalt-affinity chromatography allowed purification of LeAOS3 to >95% homogeneity (Fig. 4). The apparent molecular weight of His6-tagged LeAOS3, as determined by SDS-polyacrylamide gel electrophoresis, was in good agreement with the calculated molecular weight of 56,578. The typical yield of affinity-purified LeAOS3 was 0.5 mg/liter of E. coli culture.


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  		Fig. 4.   Affinity purification of LeAOS3 expressed in E. coli. Protein fractions obtained during the purification of His-tagged LeAOS3 were analyzed by SDS-polyacrylamide gel electrophoresis. A Coomassie Blue-stained gel is shown. Crude cell extract from E. coli cells expressing LeAOS3 was centrifuged at 100,000 × g. The resulting supernatant (lane 2) and Triton X-100R-solubilized pellet (lane 3) were analyzed. Following centrifugation of the solubilized protein at 100,000 × g, the supernatant (lane 4) was applied to a cobalt affinity column. Fractions obtained after elution of the column with 150 mM imidazole were also analyzed (lanes 5 and 6). Protein standards (lane 1) and their corresponding mass (kDa) are shown on the left.

A spectrophotometric assay (27) was used to determine whether purified LeAOS3 could metabolize fatty acid hydroperoxides (Fig. 5A). The results showed that LeAOS3 was highly active against 9-HPOD. The enzyme also metabolized 13-hydroperoxy linoleic acid (13-HPOD), albeit at a rate ~12-fold lower than that observed for 9-HPOD. A similar preference for 9-hydroperoxy linolenic acid (9-HPOT) compared with 13-HPOT was also observed (not shown; see below). These results indicate that LeAOS3 is active against both 13- and 9-hydroperoxy fatty acids, but with a marked preference for the latter substrates.


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  		Fig. 5.   Metabolism of fatty acid hydroperoxides by LeAOS3. A, utilization of 9-HPOD (solid line) and 13-HPOD (dotted line) by recombinant LeAOS3 was measured using spectrophotometric assay that monitors loss of absorbance of the substrate at 234 nm. For each assay, 250 ng of pure LeAOS3 was added to 5 µg of substrate. 9-HPOD was not metabolized by protein extracted from E. coli cells harboring the empty expression vector (dashed line). B, UV spectrum of 9-HPOD before (solid line) and after (dashed line) a 5-min incubation with recombinant LeAOS3.

The UV spectra of the reaction product generated from 9-HPOD (Fig. 5B) and other hydroperoxides (data not shown) were featureless, indicating that the substrate (lambda max = 234 nm) was largely consumed during the reaction, and that divinyl ethers (lambda max = 250-253 nm) were not among the major products. LeAOS3 also showed no detectable HPL activity using an NADH-coupled assay (36) that readily detected products formed by the action of recombinant tomato HPL on 13-HPOT (data not shown and Ref. 13). These results suggested that LeAOS3 possess neither DES nor HPL activity. To determine the enzymatic identity of LeAOS3, products generated from various substrates were converted to the corresponding methyl ester/TMS derivatives and subjected to GC-MS analysis (Fig. 6 and Table I). The major product (77.2%) obtained from reaction with 9-HPOD gave a mass spectrum that was identical to the reported spectrum (20) for the alpha -ketol, 9-hydroxy-10-oxo-12-octadecenoic acid (Fig. 6A). The second most abundant (8.7%) product gave a mass spectrum identical to that reported (18) for the cyclopentenone 10-OPEA (Fig. 6B). A third product (Fig. 6C), present in minor amounts (1.9%), was identified as the gamma -ketol (10-oxo-13-hydroxy-11-octadecenoic acid) by comparison to published spectra of this compound (e.g. Ref. 20). Because each of these products is known to arise from spontaneous hydrolysis of 9,10-EOD that is generated by the action of AOS on 9-HPOD (18, 20), we conclude that LeAOS3 is an AOS. Analysis of products derived from other hydroperoxy fatty acids gave results that were consistent with the identification of LeAOS3 as an AOS (Table I). For instance, when LeAOS3 was incubated with 9-HPOT, the reaction product contained the corresponding alpha -ketol (9-hydroxy-10-oxo-12-15-octadecadienoic acid, 61.1%), gamma -ketol (10-oxo-13-hydroxy-11-15-octadecadienoic acid, 1.8%), and the cyclopentenone 10-OPDA (3.5%). Upon incubation of LeAOS3 with 13-HPOD or 13-HPOT, the major products of catalysis were the corresponding alpha -ketol and cyclopentenone oxylipins.


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  		Fig. 6.   Mass spectra of products formed by incubation of LeAOS3 with 9-HPOD. Purified recombinant LeAOS3 was incubated with 9-HPOD at 25 °C for 5 min at pH 7.0. The resulting products were analyzed as their methyl ester TMS derivatives by GC-MS. Shown are the spectra for the alpha -ketol (9-hydroxy-10-oxo-12-octadecenoic acid) (A), the cyclopentenone 10-oxo-11-phytoenoic acid (10-OPEA) (B), and the gamma -ketol (10-oxo-13-hydroxy-11-octadecenoic acid) (C).

                              
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Table I
Analysis of LeAOS3 reaction products by GC-MS
Purified recombinant LeAOS3 was incubated with 250 µg of the indicated substrate at 25 °C for 5 min. The reaction products were analyzed as their TMS/methyl ester derivatives by gas chromatography-mass spectrometry.

The UV-visible spectrum of purified LeAOS3 was typical of the low spin ferric state, and showed a main Soret band at 418 nm (Fig. 7). Reduction of the protein with sodium dithionite and treatment with CO resulted in the appearance of a spectral peak (448 nm) that is a hallmark of cytochromes P-450. Similar spectra have been observed for other CYP74 P-450s including flaxseed AOS, melon HPL, and tomato DES (5, 23, 34). The persistence of the 420-nm peak in the LeAOS3 difference spectrum (Fig. 7, inset) was also observed in difference spectra recorded on membranes from LeAOS3-expressing E. coli cells (data not shown). These results suggest that the P-420 species does not result from inactivation of the enzyme by a step (e.g. elution with imidazole) during affinity purification. Rather, the P-420 form may reflect improperly folded protein or the weak interaction of CO with the active site of CYP74 P-450s (4, 37).


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  		Fig. 7.   Carbon monoxide difference spectrum of LeAOS3. All spectra were recorded using 100 µg of purified LeAOS3 in 1 ml of 0.1 M sodium phosphate buffer (pH 7.0). The spectra shown are for the native protein (solid line), dithionite-reduced protein (dotted line), and reduced protein bubbled with CO for 1 min (dashed line). The inset shows the difference spectrum obtained by subtracting the reduced protein spectrum from the CO-treated protein spectrum.

Purified LeAOS3 was used to determine the kinetic parameters of reactions conducted with 9-HPOD, 9-HPOT, 13-HPOD, and 13-HPOT (Table II). The apparent Km of all four substrates ranged between 4 µM (13-HPOT) and 21 µM (9-HPOD). Km values for 9-hydroperoxides were 2-4-fold higher than those for the corresponding 13-hydroperoxides. LeAOS3 was most active against 9-HPOD, as determined both by the estimated turnover rate (kcat) and catalytic efficiency (kcat/Km). The estimated kcat value (820 s-1) for 9-HPOD was comparable to turnover rates reported for other recombinant CYP74 enzymes (4, 23, 33, 34).

                              
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Table II
Substrate specificity of recombinant LeAOS3
Reactions were performed at 30 °C in 1 ml of 100 mM sodium phosphate (pH 7.0). Assays using 9-hydroperoxy fatty acids as substrate contained 30 ng of purified protein. Assays using 13-hydroperoxy fatty acids as substrate contained 60 ng of purified protein. Values represent the average ± S.D. of two experiments using independent protein preparations.

Chromosomal Location and Developmental Expression of LeAOS3-- Tomato introgression lines (ILs) were used to map LeAOS3, a single copy gene, to a region on chromosome ten that is flanked by RFLP markers CT113C and TG408 on the tomato RFLP map (data not shown and Ref. 38). EST sequencing data (www.tigr.org/tdb/lgi/) indicated that all ESTs corresponding to LeAOS3 were identified in cDNA libraries constructed from either germinating seedlings (9 of 10 ESTs) or roots (1 of 10 ESTs). To further investigate the developmental expression of LeAOS3, RNA blot analysis was used to determine the abundance of LeAOS3 transcript in various tomato organs. The results showed that LeAOS3 mRNA accumulated in roots of mature plants, with no expression detected in aerial tissues including cotyledons, leaves, stems, and flower buds (Fig. 8A). We also confirmed that LeAOS3 was expressed early after seed germination (4 days after seed imbibition), when the radical had just emerged from the seed coat (data not shown). These findings indicate that LeAOS3 transcript accumulation is tightly regulated by developmental cues and further suggest that expression of the gene is restricted to soil-exposed tissues.


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  		Fig. 8.   Developmental expression of LeAOS3. A, total RNA was extracted from unopened flower buds (B) of six-week-old tomato plants, and from root (R), stem (S), petiole (P), cotyledon (C), and leaf (L) tissue from 18-day-old plants. 5-µg samples of RNA were subjected to RNA blotting and hybridization to a LeAOS3 cDNA probe. The autoradiograph of the blot is shown together with a photograph of an ethidium bromide-stained gel of the same RNA samples (EtBr). B, total RNA was extracted from roots of 3- and 8-week-old wild-type (wt) and jai1 plants and analyzed by RNA gel blot hybridization for the presence of LeAOS3 transcripts. As a loading control, a duplicate blot was hybridized to a probe for translation initiation factor eIF4A.

Wound-induced endogenous or exogenous JAs have been shown to up-regulate the expression of AOS genes in leaves of several plants (13, 14, 39-41). To determine whether JA signaling is involved in the regulation of LeAOS3, we compared the steady-state level of LeAOS3 mRNA in roots of wild-type plants to that in the jai1 mutant that is defective in the JA response pathway (21, 42) (Fig. 8B). In contrast to the abundance of LeAOS3 mRNA in wild-type roots, expression of LeAOS3 was undetectable in roots of jai1 plants. Additional experiments showed that LeAOS3 was not expressed in wounded or JA-treated leaves of either wild-type or mutant plants (data not shown). These results show that the signaling pathway defined by jai1 regulates the root-specific expression of LeAOS3.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study we report the functional characterization of a novel tomato cDNA (LeAOS3) encoding an AOS that catalyzes the production of ketol and cyclopentenone oxylipins from both 9- and 13-hydroperoxy fatty acids. Several features of the enzyme are distinct from previously characterized AOSs involved in JA biosynthesis. First, the deduced amino acid sequence of LeAOS3 is more similar to cucumber and melon HPLs than it is to 13-AOSs from tomato (i.e. LeAOS1 and LeAOS2) or other plants. In this context, LeAOS3 represents the first example of an AOS that is classified as a CYP74C; all other AOSs are classified as CYP74As. Second, in contrast to the specificity of most AOSs for 13-hydroperoxides, LeAOS3 exhibits a marked preference for 9-hydroperoxides. A similar substrate preference was reported for the two cucurbit HPLs that are the closest known relatives of LeAOS3 (33, 34). This observation suggests that the sequence relatedness between various CYP74 P-450s is an indicator of substrate specificity. Consistent with this notion, CYP74Cs are more closely related to the 9-hydroperoxide-specific CYP74Ds than they are to members of the CYP74A and B subfamilies, which have relative specificity for 13-hydroperoxides. A third unique feature of LeAOS3 is its tissue-specific expression pattern in germinating seeds and roots of mature plants. This finding supports previous studies showing that cell-free extracts from tomato roots catalyze the formation of alpha -ketols via the 9-LOX/AOS pathway (19). Taken together, our results strongly suggest that LeAOS3 defines a class of AOSs that is distinct from those involved in JA biosynthesis in photosynthetic tissues. This idea is consistent with the notion that oxylipin metabolism is organized into 9-LOX and 13-LOX pathways, each of which use a specialized type of AOS for production of distinct oxylipins in specific cell and tissue types (3).

The overall characteristics of LeAOS3 suggest a biochemical function in the production of one or more 9-hydroperoxide-derived oxylipins. Given the expression of LeAOS3 in roots, it is conceivable that the enzyme plays a defensive role against soil-borne pests that affect roots or juvenile tissues (e.g. radical) as they emerge from the germinating seed. This hypothesis is in keeping with increasing evidence for a role of the 9-LOX pathway in plant defense against pathogens (35, 43-47). However, whereas pathogen-induced stimulation of the 9-LOX pathway has been shown to activate the DES, epoxy alcohol synthase, and reductase branches of the 9-LOX pathway (47), there is little evidence that biotic stress activates the 9-AOS pathway. A second possibility is that LeAOS3 catalyzes the production of oxylipins that play a role in plant development. Support for this hypothesis comes from two recent studies. One study provided evidence that 9-hydroxy-10-oxo-12,15-octadecadienoic acid, the alpha -ketol produced by the action of LeAOS3 on 9-HPOT, functions as a signal for flower development in Lemna paucicostata (48). Another study showed that transgenic potato plants depleted in the expression of a 9-LOX gene exhibited abnormal tuber development (49). Although specific oxylipins that account for the tuber phenotype were not identified, the presence of 9-AOS activity in potato stolons (18) is consistent with a role for 9-hydroperoxide-derived compounds in tuber development.

One of the more interesting features of LeAOS3 is its involvement in the formation of 10-OPEA and 10-OPDA from 9-hydroperoxides of linoleic and linolenic acids, respectively. These novel cyclopentenones were recently identified as products of the 9-LOX/AOS pathway in potato (18). Indeed, several similarities between LeAOS3 and the potato stolon 9-AOS activity strongly suggest that the enzymes are functionally equivalent. First, the relative abundance of 10-OPEA, alpha -ketol, and gamma -ketol products formed by the action of LeAOS3 on 9-HPOD was comparable to that reported for the potato enzyme. Second, the tissue-specific expression of LeAOS3 is similar to that of the cyclopentenone-forming activity in potato, which was highest in roots and not detectable in leaves. A third similarity between LeAOS3 and the potato 9-AOS was the relatively high proportion of cyclopentenone product (i.e. 10-OPEA) formed from 9-HPOD. This feature of 9-AOS-catalyzed metabolism of hydroperoxy dienoic fatty acids contrasts other studies showing that non-enzymatic cyclization of allene oxides requires the presence of a double bond in the beta ,gamma -position relative to the epoxide group (50-52).

The formation of cyclopentenones by LeAOS3 is particularly interesting in light of the signaling activity exhibited by cyclopentenones (e.g. 12-OPDA) produced from the 13-LOX/AOS pathway. Recent studies (53) in Arabidopsis have shown that 12-OPDA can activate the expression of defense-related genes via a signal transduction pathway involving COI1, an F-box protein that is required for JA-mediated signaling (54). There is also evidence to suggest that the alpha ,beta -unsaturated carbonyl group located in the cyclopentenone ring 12-OPDA is involved in defense gene activation via a COI1-independent signaling pathway (53). Such a signaling mechanism has been documented for cyclopentenone prostaglandins in which the reactive alpha ,beta -unsaturated carbonyl mediates conjugate addition to various intracellular targets (55). Given the structural similarity of 9-AOS-derived cyclopentenones to 12-OPDA (Fig. 1), including the presence of an alpha ,beta -unsaturated carbonyl group in the cyclopentenone ring, a role for these compounds in signaling can be hypothesized. Additional work is needed to determine whether 9-AOS catalyzes the formation of 10-OPEA and 10-OPDA in plant tissues.

We observed that LeAOS3 mRNA accumulation in roots of mature plants is dependent on the Jai1 gene product that is a component of the jasmonate signaling pathway in tomato. This finding suggests that the 9-LOX/AOS pathway in tomato roots is tightly regulated by JA or a related compound that signals through Jai1. Support for this idea comes from experiments showing that LeAOS3 expression is significantly reduced in roots of the def1 JA biosynthetic mutant.3 The dependence of root-specific LeAOS3 expression on Jai1 may provide clues to the biological function of the enzyme. For example, JA signaling has been shown to play an important role in resistance of Arabidopsis to soil-borne pathogens (56, 57), presumably as a result of activation of defensive processes in roots. It is thus conceivable that the 9-LOX/AOS pathway is a component of a JA-regulated defense system against soil-borne pathogens or other pests that attack root tissues. The lack of LeAOS3 expression in jai1 roots may provide a convenient experimental system in which to address this possibility, and to further investigate the interaction between the 9- and 13-LOX pathways.

    ACKNOWLEDGEMENTS

We thank Beverly Chamberlin and Doug Gage of the MSU Mass Spectrometry Facility for assistance with mass spectrometry. We also thank Dr. David Nelson for making the CYP74C assignment. Tomato EST clones cLED1D24 and cLEI16I12 were obtained from the Clemson University Genomics Institute.

    FOOTNOTES

* This work was supported by grants from the Division of Energy Biosciences, the United States Department of Energy (DE-FG02-91ER20021), and Michigan Life Science Corridor (78341) (to G. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF454634.

§ These authors contributed equally to this work.

Present address: Dept. of Environmental Information and Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.

** To whom correspondence should be addressed: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312. Tel.: 517-355-5159; Fax: 517-353-9168; E-mail: howeg@msu.edu.

Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M207234200

2 D. Nelson, personal communication.

3 A. Itoh and G. Howe, unpublished results.

    ABBREVIATIONS

The abbreviations used are: LOX, lipoxygenase; AOS, allene oxide synthase; HPL, fatty acid hydroperoxide lyase; AOC, allene oxide cyclase; DES, divinyl ether synthase; OPR, 12-OPDA reductase; JA, jasmonic acid; MeJA, methyl JA; EST, expressed sequence tag; RT-PCR, reverse-transcriptase-PCR; TAIL-PCR, thermal asymmetric interlaced-PCR; GC-MS, gas chromatography-mass spectrometry; RFLP, restriction fragment length polymorphism; 9-HPOD, 9-hydroperoxy linoleic acid; 9-HPOT, 9-hydroperoxy linolenic acid; 13-HPOD, 13-hydroperoxy linoleic acid; 13-HPOT, 13-hydroperoxy linolenic acid; 9, 10-EOT, 9,10-epoxyoctadecatrienoic acid; 9, 10-EOD, 9,10-epoxyoctadecadienoic acid; 12, 13-EOT, 12,13-epoxyoctadecatrienoic acid; 10-OPDA, 10-oxo-11,15-phytodienoic acid; 10-OPEA, 10-oxo-11-phytoenoic acid; 12-OPDA, 12-oxo-10,15-phytodienoic acid; TMS, trimethylsilyl.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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