This document summarizes research on a novel lipoxygenase enzyme found in pea roots called LOXN2. Key findings include: 1) LOXN2 was cloned from pea and shown to encode a 93.7 kD protein with two deletions compared to other plant lipoxygenases. 2) When expressed in yeast, LOXN2 exhibited lipoxygenase enzyme activity, preferentially oxygenating linoleic acid to produce both 9- and 13- hydroperoxy octadecadienoic acids in a 3:1 ratio. 3) LOXN2 transcription was found to be downregulated in pea roots infected with the cyst nematode Heterodera goet

1. A Novel Lipoxygenase in Pea Roots. Its Function
in Wounding and Biotic Stress
Pasqua Veronico*, Donato Giannino, M. Teresa Melillo, Antonella Leone, Aurelio Reyes,
Malcolm W. Kennedy, and Teresa Bleve-Zacheo
Institute of Plant Protection, Bari Section, Consiglio Nazionale delle Ricerche, 70126 Bari, Italy (P.V., M.T.M.,
T.B.-Z.); Institute of Biology and Agricultural Biotechnology, Rome Section, Consiglio Nazionale delle
Ricerche, 00016, Monterotondo, Rome, Italy (D.G.); Institute of Science of Food Production, Lecce Section,
Consiglio Nazionale delle Ricerche, 73100 Lecce, Italy (A.L.); Dunn Human Nutrition Unit, Medical Research
Council, Cambridge CB2 2XY, United Kingdom (A.R.); and Division of Environmental and Evolutionary Biology,
Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom (M.W.K.)
The genome of pea (Pisum sativum) contains genes encoding a family of distinct lipoxygenases (LOX). Among these, LOXN2
showed eight exons encoding a 93.7-kD enzyme, harboring two C-terminal deletions and an unusual arginine/threonine-
tyrosine motif in the domain considered to control the substrate specificity. LOXN2, when overexpressed in yeast, exhibited
normal enzyme activity with an optimum at pH 4.5, and a dual positional specificity by releasing a 3:1 ratio of C-9 and C-13
oxidized products. The predicted LOXN2 structure lacked a loop present in soybean (Glycine max) LOX1, in a position
consistent with control of the degree of substrate access to the catalytic site and for LOXN2’s dual positional specificity. The
LOXN2 gene was tightly conserved in the Progress 9 and MG103738 genotypes, respectively, susceptible and resistant to the
root cyst nematode Heterodera goettingiana. LOXN2 transcription was monitored in roots after mechanical injury and during
nematode infection. The message peaked at 3 and 24 h after wounding in both genotypes and was more abundant in the
resistant than in the susceptible pea. In nematode-infected roots, transcription of several LOX genes was triggered except
LOXN2, which was repressed in both genotypes. In situ hybridization revealed that LOXN2 message was widespread in the
cortex and endodermis of healthy roots, but specifically localized at high level in the cells bordering the nematode-induced
syncytia of infected roots. However, LOXN2 transcript signal was particularly intense in collapsing syncytia of MG103738
roots, suggesting LOXN2 involvement in late mechanisms of host resistance.
Lipoxygenases (LOX; linoleate:oxygen reductase, 1996). The transcription of each gene member is under
E.C. 1.13.11.12) are nonheme iron-containing enzymes tight developmental control, and more than one mem-
that catalyze the addition of molecular oxygen at ber is often active at a specific developmental stage,
either the C-9 or C-13 residue of fatty acids with a accounting for the occurrence of multiple LOX iso-
1,4-pentadiene structure. Linoleic and linolenic acids forms. These exhibit distinct features for preference of
are the most abundant fatty acids in the lipid fraction substrate, kinetic parameters, and positional specificity
of plant membranes and are the major substrates for of substrate oxygenation (Feussner and Wasternack,
LOXs. The oxygenation step leads to a reaction cas- 2002). In pea (Pisum sativum), LOX genes exhibit
cade (termed the LOX pathway), in which the hydro- tissue specificity and are developmentally regulated
peroxides (HPOs), produced by the LOX activity, are (Domoney et al., 1990). A few of them have been
substrates of HPO lyases and allene oxide synthases proposed to be involved in the development of root
(Vick, 1993). HPOs are highly reactive and rapidly de- nodules (Wisniewski et al., 1999) and to play a defen-
grade into metabolites that are precursors for jasmonic sive role in trophic relationships between plants and
acid, methyl jasmonate, conjugated dienoic acids, and pathogenic nematodes (Leone et al., 2001).
volatile aldehydes. These products are known to play Phytoparasitic nematodes are the most widespread
a role in plant defense (Wasternack et al., 1998). and deleterious pests for many crops. Several complex
LOXs are encoded by gene families (LOX) in most, if mechanisms, which underlie the interaction between
not all, of the plant species studied so far (Royo et al., the plant and endoparasitic pathogen, induce the
differentiation of host cells into feeding structures nec-
essary for parasite development. In particular, cyst
* Corresponding author; e-mail p.veronico@ba.ipp.cnr.it; fax: 39–
nematodes enter the root and migrate to the vascular
080–558–0468.
The author responsible for distribution of materials integral to the
cylinder where they select a cell that becomes the
findings presented in this article in accordance with the policy initial feeding site. Once the parasite has established,
described in the Instructions for Authors (www.plantphysiol.org) is: this cell is induced to develop into a syncytium as the
Pasqua Veronico (p.veronico@ba.ipp.cnr.it). result of cell-wall breakdown and subsequent fusion
Article, publication date, and citation information can be found at of neighboring cell protoplasm. A particularly serious
www.plantphysiol.org/cgi/doi/10.1104/pp.106.081679. pest of pea is the cyst nematode Heterodera goettingiana,
Plant Physiology, July 2006, Vol. 141, pp. 1045–1055, www.plantphysiol.org Ó 2006 American Society of Plant Biologists 1045

2. Veronico et al.
which is difficult to control due to its long soil persis- AAB71759), LOXN4 (EMBL accession no. CAC04380),
tence and resistance to chemical control (Thompson and LOXN5 (EMBL accession no. CAA75609), respec-
et al., 2000). In this context, the isolation of genes in- tively. LOXN2 contained all the functional domains
volved in conferring host resistance represents a poten- typical of LOXs (Fig. 1): an N-terminal region forming
tially valuable approach to improving crop breeding a b-barrel structure (Boyington et al., 1997), shown
programs. to target lipid body LOX to liposomes and lipid bod-
In the past decade, a considerable number of pea ies (May et al., 2000), and the catalytic site in the
genes responsible for resistance against a range of C-terminal domain. Moreover, a search for intracellu-
pathogens, but not nematodes, have been character- lar sorting and processing peptides in the predicted
ized (Chang et al., 1995; Culley et al., 1995; Pilet-Nayel amino acid sequence of LOXN2 (pSORT program;
et al., 2002; Prioul et al., 2004; Timmerman-Vaughan http://psort.ims.u-tokyo.ac.jp/) suggested that LOXN2
et al., 2004). In addition, sources of resistance to H. is probably confined to the cytosol because it con-
goettingiana have been found in the germplasm tained neither consensus targeting nor retention sig-
MG103738 accession of pea (Di Vito and Perrino, nals for any organelles.
1978). Moreover, it has been reported that the nema- A stretch of approximately 50 amino acids (positions
tode was able to infect MG103738 roots and to induce 1–52) in the N-terminal region differed from those of
syncytia, but they degenerated very rapidly (Bleve- other pea LOXs. However, a high degree of similarity
Zacheo et al., 1990; Melillo et al., 1990). The degrada- was observed in the following region, spanning over
tion of trophic cells, indicative of a hypersensitive 100 residues, which is predicted to be a b-barrel
response (HR), was accompanied by an increased structure by standard secondary structure analysis
activity of LOXs (Zacheo et al., 1997). The LOX activity programs (e.g. National Center for Biotechnology In-
was triggered during nematode infection in the sus- formation conserved domain search) and three-
ceptible genotype (Progress 9) and in the resistant dimensional molecular modeling (see below). In the
MG103738, although the levels were remarkably higher C-terminal region, LOXN2 has two deletions of six and
in the resistant than in the susceptible roots. In 27 amino acids as compared to the stretches 277 to 281
MG103738, the highest level of LOX activity coincided and 296 to 322 of the soybean (Glycine max) LOX1
with the occurrence of collapsed syncytia and necro- (Swiss-Prot accession no. P08170), which is one of the
tizing cells (48 h after nematode infection), whereas in best characterized among LOXs. A molecular model of
Progress 9 it occurred later during syncytial develop- LOXN2 was produced by homology modeling using
ment and was accompanied by a weak increase of five templates for crystal structures of LOX proteins
HPOs (Leone et al., 2001). from another legume, soybean (Fig. 2).
This study focused on the molecular cloning and The deletions located in the domain III (Fig. 1) lead
characterization of LOXN2, a member of the pea LOX to the absence of an external loop, but did not change
family genes, during plant-nematode interaction. the position and conformation of the b-barrel and the
LOXN2 represented a novel isoform in the pea able catalytic site of LOXN2 (Fig. 2). The residues involved
to produce both C-9 and C-13 HPOs. The gene tran- in iron binding (His-486, His-491, His-677, Asn-688,
scription was down-regulated upon nematode infec- and Ile-826) were conserved in domain V. The posi-
tion in the resistant genotype, exhibiting an inverse tional specificity of plant LOXs is usually determined
trend to that of other LOX family members. Moreover, by the motifs R/TH or R/TF for 13-LOX and R/TV for
the message localization in the resistant and suscepti- 9-LOX in the active site (Feussner and Wasternack,
ble genotypes leads us to hypothesize a role for 2002). Unusually, LOXN2 contained a Thr-Tyr motif
LOXN2 in the antinematode defense system. (R/TY; Fig. 1), which has not been described previ-
ously to our knowledge.
A phylogenetic tree (Fig. 3) was constructed based on
gap-free multi-alignments of dicot and monocot LOX
RESULTS sequences currently available. Pea LOXN2 fell into the
The LOXN2 cDNA, Encoded Protein, highly supported monophyletic group of leguminous
and Protein Model Structure species. However, its closest relative was not the other
previously identified pea LOX (LOXN5) but lentil (Lens
The full-length cDNA of LOXN2 (EMBL accession culinaris) LOX (0.22 substitutions/site). Indeed, within
no. AJ749702) harbored an open reading frame (ORF) leguminous species, two well-supported clusters were
of 2,481 nt encoding a presumptive translation product found, each containing a different pea LOX sequence.
of 826 amino acids with a Mr of 93,787 and a pI of 5.18 Interestingly, none of the closely related LOX showed
(ProtParam tool: Wilkins et al., 1998). The ORF was the deletions described for the pea LOXN2.
flanked by a 21-nucleotide (nt) 5#-untranslated region
(UTR) and a 148-nt 3#-UTR containing a canonical Biochemical Activity of Recombinant Yeast LOXN2
polyadenylation signal.
The predicted protein product LOXN2 showed To study the biochemical function and to test
57.4%, 57.1%, and 58.2% identity and 84.4%, 85.6%, and whether the lack of a loop in the C terminus affected
84.9% similarity to pea LOXN1 (EMBL accession no. its activity, LOXN2 was cloned in the expression vector
1046 Plant Physiol. Vol. 141, 2006

3. A New Pea Lipoxygenase Involved in Abiotic and Biotic Stress
Figure 1. Alignment of the deduced amino acid sequence of pea LOXN2. Shown is protein sequence alignment of pea LOXN2
(EMBL accession no. AJ749702) with pea LOXN1 (GenBank accession no. U84198), LOXN4 (GenBank accession no.
AJ293015), LOXN5 (GenBank accession no. Y15410), and soybean LOX1 (GenBank accession no. J02795). Gaps introduced for
better alignment are shown by dashes, and asterisks indicate residues involved in binding of the iron atom. Black shading
corresponds to identical amino acid residues. Presumed motifs responsible for positional specificities are shown by gray shading.
Domains as assigned by Minor et al. (1996) are underlined.
pPIC9 and over-expressed in Pichia pastoris (strain Plant LOXs are usually classified as 9-LOXs and
GS115). In induced strains (see ‘‘Materials and 13-LOXs on the basis of their product specificities. As
Methods’’), LOXN2 was represented by a strong band pea LOXN2 was found to contain the unusual R/TY
of expected size (approximately 94 kD) in both silver- motif in the active site, its positional specificity was
stained polyacrylamide gels and western blots, tested. Linoleic acid was selected as substrate in 0.1 M
whereas no signal was revealed in control strains har- sodium acetate buffer at pH 4.5 and 0.1 M sodium
boring the empty vector (Fig. 4). phosphate buffer at pH 6.0. The reaction products
The biochemical activity of recombinant LOXN2 were reduced with sodium borohydride and sepa-
was investigated in extracts of yeast expressing the rated by reverse-phase HPLC. The peak adsorbing at
protein (Table I) by measuring the increase of A234 234 nm and containing the HPOs was collected and
using linoleic, linolenic, and arachidonic acids as sub- subjected to straight-phase HPLC to separate 13- and
strates. The optimum pH was determined for each sub- 9-hydroxy octadecadienoic acid isomer (HODE). The
strate by varying the pH of the reaction buffers. The retention times of LOXN2 reaction products were
activity of LOXN2 was detected with all the substrates consistent with both authentic standards of 9- and
in a range of pH 4.5 to 8.0. In particular, at pH 4.5, the 13-HODE and those of soybean LOX1. In the presence
maximum activity of 1.35 units/mg protein occurred of both buffers, LOXN2 produced 9- and 13-HODE in a
with linoleic acid, whereas it fell to 0.23 units/mg 3:1 ratio (Table I).
protein and 0.18 units/mg protein with linolenic and
arachidonic acids, respectively. However, at pH 6.0, Genomic Organization
the maximum activity was 0.45 units/mg protein with
arachidonic acid, followed by 0.18 units/mg protein Genomic DNA of MG103738 and Progress 9 was
and 0.07 units/mg protein with linolenic and linoleic endonuclease restricted, size fractionated, transferred
acids, respectively. The extracts from controls did not onto a nylon membrane, and hybridized with a di-
show any activity. goxigenin (DIG)-labeled genomic DNA probe spanning
Plant Physiol. Vol. 141, 2006 1047

4. Veronico et al.
LOXN2 transcript detected by semiquantitative re-
verse transcription (RT)-PCR (Fig. 6, A and B). In
unwounded plants, the variation of LOXN2 expres-
sion appeared to be constant and the message abun-
dance at 1 h was recorded as control. In both genotypes
the transcript showed a peak level within 3 h, followed
by a decrease at 6 h and a further growth at 24 h after
injury. Within 3 h, the transcript in Progress 9 was
9-fold higher, whereas in MG103738 it was 15-fold
higher than in the respective controls. The response
to wounding was therefore more intense in MG103738
than in Progress 9, suggesting a different reactivity
between the two genotypes. Moreover, during the
Figure 2. Model of LOXN2 structure. Shown are a homology model of
pea LOXN2 (right) created from high-resolution crystal structures of
similar proteins from legumes, as described in ‘‘Materials and
Methods,’’ and the crystal structure of soybean LOX1 for comparison
(left). The distinctive b-rich region is highlighted at the top left of each
structure, and the external loop that is present in soybean LOX1 but
absent in pea LOXN2 is shown space-filled.
the two- to six-exon region (Fig. 5A). This probe, which
lacked 105 bp compared to other pea LOX members,
was designed for LOXN2 specificity. The resulting
pattern of hybridization (Fig. 5B) consisted of a single
band with all the restriction enzymes used (which did
not cut in the probe) and was identical in both geno-
types. The sizes of DNA fragments detected were con-
sistent with those predicted from the restriction map
of genomic sequences. These results suggest the occur-
rence of one LOXN2 copy and a very high degree of
identity in both genotypes.
To search for introns, PCR experiments were per-
formed on genomic DNA with primer combinations
designed along the full-length transcript. The se-
quence comparison confirmed that LOXN2 was iden-
tical in both genotypes and contained eight exons and
seven introns (Fig. 5A). The exon positions were
conserved with respect to pea LOXN5 (GenBank ac-
cession no. Y15410), in which the exons 6 and 7 were
separated by an intron that is absent in LOXN2 (Fig.
Figure 3. Phylogenetic tree of LOXN2. Pea LOXN2 amino acid se-
5A). Moreover, LOXN2 introns were rich in A/T nu- quence was compared to other monocot and dicot LOXs available in
cleotides (73%) and harbored the canonical splicing the EMBL database (accession nos. are shown in parentheses). Amino
GT/AG motifs. acid sequences were multialigned with ClustalW and edited within
PILEUP program where necessary (1994 GCG program manual). A
LOXN2 Expression in Response to Mechanical Injury nucleotide multialignment was also created based on the result of the
optimized amino acid multialignment. The phylogenetic relationship
Pea LOXs represent a gene family with a discrete between sequences was established using the following procedures.
polymorphism in the 3# ends among the members. Genetic distances were obtained from the multialigned nucleotide
Therefore, a specific probe was designed in the 3#-UTR sequences using the GTR method implemented in PAUP (Swofford,
(Fig. 5A) to monitor LOXN2 expression in roots under 1998). The resulting distance matrix was used for tree reconstruction
abiotic and biotic stresses. Because nematodes invade using both the neighbor-joining (NJ) and minimum evolution (ME)
approaches. In addition to distance methods, we have also applied a
host roots by cell perforation using their stylet, changes
discrete method, Maximum Parsimony (MP), to the multialigned pro-
in LOXN2 gene transcription following mechanical tein sequences using PAUP (Swofford, 1998). In all cases, monocot
injuries were examined. species were used as outgroups and bootstraps values (listed at the
The root elongation zone and apices of 1-week-old branching point) were based on 1,000 repetitions. The lengths of the
MG103738 and Progress 9 seedlings were needle branches in the tree are proportional to the genetic distance, according
punctured, collected at different time intervals, and to the scale at the bottom.
1048 Plant Physiol. Vol. 141, 2006

5. A New Pea Lipoxygenase Involved in Abiotic and Biotic Stress
(Fig. 7A), and the other was specific for LOXN2 (Fig.
7B). As for plants left uninfected, LOX levels were
observed to vary with time, suggesting a regulation
during root development. Hence, it was considered
that the comparison of transcript levels between
infected and uninfected roots, at a specific time point,
had a biological meaning in the responses to nematode
infection elicited in the different host genotypes. The
global expression level of LOXs (Fig. 7A), which did
not vary significantly between resistant and suscepti-
ble peas at 24 h, dropped in the susceptible and
increased in the resistant genotype 48 h after nematode
infection compared to uninfected roots. On the con-
trary, LOXN2 transcript (Fig. 7B), 24 and 48 h following
nematode infection, was down-regulated in both in-
fected resistant (0.6- and 0.8-fold versus control) and
susceptible roots (0.79- and 0.8-fold versus control).
Figure 4. Protein-blot analysis and SDS-PAGE of LOXN2. A, Crude
protein extracts (10 mg) from P. pastoris GS115 transformed either with
pPIC9 (control plasmid) or with pPIC9/LOXN2 were separated on SDS- Localization of LOXN2 Message upon
PAGE gels, blotted, and probed with anti-LOX antibody. B, Silver- Nematode Infection
stained SDS-PAGE gel of pPIC9 and pPIC9/LOXN2 extracted from yeast
cultures. Positions of Mr marker proteins are as indicated (kD). To determine the spatial expression pattern of
LOXN2 during nematode infection, in situ experi-
ments were conducted on serial cross sections of
decrease phase, LOXN2 transcript abundance in uninfected and 48 h infected roots. Sense and antisense
MG103738 was 10 to 8 times higher than in the control, riboprobes spanning the LOXN2 3#-UTR were used
while in Progress 9 it fell down to the level of its (Fig. 5A). In uninfected roots, the transcript was visu-
control. LOXN2 message levels in MG103738 at 24 h alized in cortical and endodermal cells as a purple
were 16-fold higher than in the control, whereas the staining, which appeared more intense in the resistant
message abundance in Progress 9 was 8 times higher than in the susceptible genotype (Fig. 8, A and B). In
than in the unwounded control samples. Taken resistant infected roots, faint and infrequent signal
together, these data suggest that LOXN2 maintains spots were observed in the cortex, but LOXN2 message
a higher grade of expression in MG103738 than in was abundant in the outer cells surrounding the vas-
Progress 9. cular cylinder, in those cells injured by nematode
penetration and those flanking the induced syncytia
Global LOX Transcription Is Triggered in the Infected (Fig. 8, C and E). In susceptible infected roots, the
Resistant Genotype, But LOXN2 Is Repressed transcript was detected in cortical cells damaged by
nematode invasion and undergoing necrosis, and in
Transcript abundance was monitored in nematode- parenchymatous cells of the vascular cylinder strictly
challenged roots of MG103738 and Progress 9 genotypes related to syncytia development (Fig. 8D). In MG103738
and compared to uninfected root tissues. Northern roots, syncytia rapidly degenerated and were highly
analyses were performed at 24 and 48 h after reactive to the probe due to the condensed cytoplasm,
H. goettingiana infection using two distinct probes which is a feature of collapse (Fig. 8, E and D). No
(Fig. 5; see also ‘‘Materials and Methods’’): one con- LOXN2 mRNA was detected in infected and unin-
tained a highly conserved stretch in the ORF and was fected root sections when hybridized with a sense
used to detect a message pool from LOX members probe (Fig. 8, G and H).
Table I. Substrate and product specificity of pea 9-/13-LOXN2
All activities were determined in 0.1 M sodium acetate buffer, pH 4.5, at 25°C with 0.3 mM substrate measuring the increase of A234. The relative rate
(%) of hydroperoxidation with respect to linoleic acid hydroperoxidation is indicated in parentheses.
Substrates
Property
Linoleic Acid (18:2) Linolenic Acid (18:3) Arachidonic Acid (20:4)
Substrate specificity
Specific activity (mmol/min per mg of protein)a 1.35 6 0.02 (100%) 0.23 6 0.04 (17%) 0.18 6 0.03 (13%)
Product specificity
Ratio of positional isomers of HPOs (13-HPOD:9-HPOD) 1:3
a
Data are mean 6 SE from three independent experiments.
Plant Physiol. Vol. 141, 2006 1049

6. Veronico et al.
The Unusual Structure of LOXN2 Does Not Affect the
Enzyme Function and Confers Dual Positional Specificity
Analysis of the LOXN2 coding sequence showed
that the predicted protein contains all of the domains
typical of LOX enzymes. However, a few peculiarities
distinguished LOXN2 from most of the plant LOXs,
which are usually organized into a small N-terminal
b-barrel and a large C-terminal catalytic domain
(Boyington et al., 1993). In contrast, Minor et al. (1996)
Figure 5. Genomic organization of LOXN2 gene. A, Schematic repre-
sentation of the pea LOXN2 gene in comparison with lox1:ps:5
(LOXN5, GenBank accession no. Y15410) gene structure. The trans-
lation start and the stop codon are indicated. Filled boxes represent the
exons (sizes are indicated), and the single line between filled boxes
represents the introns. 5#-UTR and 3#-UTR are indicated as dotted
lines. E, H, and S indicate the positions of restriction sites for EcoRI,
HindIII, and SacI, respectively. Gel blots of genomic DNA were
hybridized with probe 1. In northern blots and in situ analyses, probe
2 was used. B, Southern hybridization of pea genomic DNA with
LOXN2 gene-specific probe. Genomic DNA (8 mg) of both resistant
(MG103738) and susceptible (Progress 9) pea genotypes was digested
with EcoRI (E), HindIII (H), and SacI (S) and hybridized with a 1.1-kb
fragment. DNA size standards in kb are shown at left.
DISCUSSION
In the genome of the pea, the LOX genes encode a
family of enzymes, and those expressed in roots have
been proposed to play a role in defense mechanisms
against cyst nematodes (Leone et al., 2001). In this Figure 6. RT-PCR analysis of the expression patterns of LOXN2 in
wounded pea roots. Shown is ethidium bromide-stained agarose gel
report, we focused on the structure, the transcription
analysis of RT-PCR. cDNAs from wounded resistant (A) and susceptible
pattern, and localization of LOXN2 in response to the (B) pea roots at the time points indicated were produced and used as
cyst nematode H. goettingiana and the biochemical template. The 26S gene was used as control for amplification. The
properties of the enzyme. The unusual structural graph shows normalized values of signal intensities of the RT-PCR
features of LOXN2 are, surprisingly, not reflected in products. Expression analyses were carried out twice with independent
an alteration of the biochemical activity of the en- RNA extracts. Bars indicate SEs and the bands in the gel are the result of
zyme. one of the experiments.
1050 Plant Physiol. Vol. 141, 2006

7. A New Pea Lipoxygenase Involved in Abiotic and Biotic Stress
proposed to configure LOXs into four small domains
that associate on the surface of a large C-terminal do-
main. The latter harbors the catalytic site, whereas the
other domains are proposed to facilitate binding,
transport, and release of both substrates and products.
Modeling (Swissmodel) of pea LOXN2 against the
crystal structure of soybean LOX1 (Protein Data Bank
[PDB] accession no. 1FGM) indicated that LOXN2
lacked an external loop, lost due to two deletions of six
and 27 amino acids in the domain III. This loss did not
cause any conformational change in the b-barrel do-
main and the catalytic site of the protein. LOXN2
maintained an intact LOX-like activity after being
expressed in P. pastoris, and the optimal pH of activity
Figure 8. Localization of LOXN2 transcripts in pea roots. Shown is in
situ hybridization of LOXN2 transcript in resistant and susceptible pea
in uninfected roots (A and B) and nematode-infected roots (C–E). Root
cross sections were hybridized to DIG-labeled LOXN2 antisense (A–E)
and sense (G and H) riboprobes or stained (F) with a 0.05% toluidine
blue solution. A and B, Uninfected roots of resistant MG103738 (A) and
susceptible Progress 9 (B). The purple signal representing LOXN2
message is visible more intensely in differentiated cortex and endoder-
mal cells in resistant (A) than in susceptible (B) root. C to E, Forty-eight
hour nematode infected roots of MG103738 (C and E) and Progress
Figure 7. Gene expression in pea roots after nematode infection. 9 (D). In the resistant root, the signal intensity decreases in the cortex but
Northern blots were performed on total RNA (5 mg) from root apices is intensely spotted in endodermal cells (C). The signal is also visible in
and elongation zones of resistant (MG103738) and susceptible (Pro- the cells injured by nematode penetration and in cells surrounding the
gress 9) pea genotypes at the time points indicated (h) after nematode syncytium (E, white arrow). Syncytium in the resistant root section (C) is
infection by using a radioactively labeled probe corresponding to the less developed than in the susceptible genotype (D) and its displaying
LOX ORF (A) and a specific cDNA probe spanning LOXN2 3#-UTR (B). of transcript signaling is due to condensed necrotizing cytoplasm;
Control samples were uninfected roots (NI) from seedlings kept at the LOXN2 transcript signal in susceptible root occurs in endodermal cells
same conditions as the infected ones (I). ID Image Analysis software and in parenchyma nematode-wounded cells (D). F, Syncytia (white
(Kodak Digital Science) was used to quantify the intensity of each band. arrow) in D developed normally as confirmed by toluidine blue-stained
Relative gene expression was represented in histograms as the optical cross section. G and H, Control experiments represented by infected
density ratio between the tested gene and 26S rRNA. The experiment and uninfected resistant roots hybridized with the sense DIG-LOXN2
was repeated three times, and the results illustrated are typical of three probe. ne, Nematode. A to D and G and H, bars 5 2.5 mm; E and F,
separate experiments. SEs are indicated by size bars. bars 5 1.6 mm.
Plant Physiol. Vol. 141, 2006 1051

8. Veronico et al.
had a much lower value (4.5) than that of other pea sponse to wounding was remarkably rapid; maximum
LOXs (Hughes et al., 1998). Other LOXs with acidic pH transcript levels were reached within 3 h after treat-
optima have been described in rice (Oryza sativa; Ida ment and they were transient and biphasic. The resis-
et al., 1983), soybean (Baracat-Pereira et al., 2001), tant genotype reacted more strongly than the
carnation (Dianthus caryophyllus; Rouet-Mayer et al., susceptible genotype in increasing LOXN2 expression
1992), and rose (Rosa hybrida; Fukuchi-Mizutani et al., to wounding. A similar kinetic expression of LOX with
2000). A role of acidic LOXs in the senescence of DPS has been reported in maize (Zea mays) in response
carnation and rose flowers has been suggested to be to exogenous methyl jasmonic acid treatment (Kim
the result of membrane disruption by the HPO and et al., 2003). These authors suggested that the activa-
free radical action (Rouet-Mayer et al., 1992; Fukuchi- tion of LOX expression in the early phase of a wound
Mizutani et al., 2000). response involved 13-LOX activity that was in turn
Plant LOXs are classified into 9- and 13-LOXs ac- followed by its 9-LOX activity at the late phase.
cording to the position at which the oxygenation of Together with these previous findings, our results sug-
linoleic acid occurs. Most of the plant LOXs harbors a gest that LOXN2 expression in pea genotypes might
tightly conserved Arg in the substrate-binding pocket. exhibit a similar behavior to nonconventional maize
In 13-LOXs of several plants, the occurrence of His or LOX. LOXN2 seems to be rapidly involved in the
Phe has been proposed to mask the positive charge of oxidative burst elicited by mechanical cell damage,
Arg and to hamper the interaction with the linoleate thereafter coming back to its normal function in tissue
carboxylate. This should favor the entry of the linoleate development. In pea, LOXN2 transcript levels were
methyl terminus and the C-13 oxidation (Hornung et al., higher in resistant than in susceptible genotypes at all
1999). In contrast, in 9-LOXs the His and Phe are time points tested. The exon-intron structure, the
replaced by a Val, which is thought thereby to allow sequence, and the copy number (one) of LOXN2 were
the inverse orientation of linoleate (Feussner and identical in both genotypes. Therefore, the difference
Wasternack, 2002). However, the positional specificities of expression levels is likely due to divergences in
of some plant LOXs (e.g. those of monocots) do not fit transcription control mechanisms (e.g. distinct epige-
this predictive model and a class of nonconventional netic factors, promoter/enhancer sequences, repres-
LOXs was therefore proposed (Feussner and Wasternack, sors, signal transducers, etc.).
2002). Moreover, Hughes et al. (2001) reported that the Following nematode infection, the abundance of
mechanisms controlling dual positional specificity mRNA representing root LOXs increased in the resis-
(DPS; Hughes et al., 1998) in pea 9-/13-LOX and tant (but not in the susceptible) genotype while mRNA
cucumber (Cucumis sativus) 13-/9-LOX were different. of LOXN2 decreased, indicating that LOX family mem-
DPS of pea LOX was more likely to be determined by bers responded differentially to the infection. Triggering
the degree of penetration of the methyl terminus and of plant LOX gene transcription is the most frequently
the volume of the linoleate binding pocket rather than observed response to pathogen attacks (Porta and
substrate orientation. As for pea LOXN2, the common Rocha-Sosa, 2002). However, there is a body of evi-
His, Phe, or Val residues were replaced by a Tyr, which dence that some LOXs, together with other defense
is without precedent among plant LOXs. Tyr is of a genes, decrease their expression during infection (Moy
similar size to His and Phe, and this might lead to a 13- et al., 2004). Down-regulation of LOXN2 occurred at
LOX activity. Surprisingly, LOXN2 showed a DPS by 24 h after nematode inoculation and maintained the
releasing 9- and 13-HPOs in a 3:1 ratio. DPS was also same trend until 48 h, when the parasite induces the
observed in pea LOX-2 and LOX-3 (Hughes et al., 1998), formation of syncytial cells. Interestingly, LOXN2
but they retain the Phe residues. Consequently, our mRNA levels dropped sharply in the resistant geno-
findings are consistent with the hypothesis that the type, suggesting again that distinct mechanisms of
conformation of domain III of LOXN2 may have a role transcriptional regulation occur for the two LOXN2
in determining DPS by controlling the degree of lino- paralogs.
leate access into the catalytic site, in agreement with In uninfected peas, the occurrence of LOXN2 tran-
Hughes et al. (2001). Finally, the LOX specificity has script in cortical and endodermal cells suggests a central
implications for HPO metabolism; 13-HPO of linolenic function for this enzyme in root development. In light of
acid is converted into jasmonate by allene oxide syn- these results, it can be hypothesized that LOXN2 with
thase, whereas the HPO lyase acts on 9- and 13-HPOs to acidic pH optimum, as for other proteins like expansins,
form volatile aldehydes. In this regard, the DPS of could mediate relaxation of cell walls during acid-
LOXN2 suggests that it is able to provide compounds induced growth (McQueen-Mason et al., 1992). In in-
that play a role in both developmental process and fected roots, LOXN2 message was mostly detected in the
defense response (Siedow, 1991). cells bordering the syncytia. The abrupt loss of signal in
the cortical tissue, accompanied by its confinement to the
LOXN2 Response to Wounding and Nematode Infection few cells delimiting the syncytium, was consistent with
the expression dropping, as revealed by northern anal-
Accumulation of LOXN2 transcripts over their nor- yses in infected roots. It is worth noting that resistant and
mal basal level was induced by wounding in both susceptible genotypes shared a similar pattern of tran-
resistant and susceptible genotypes. The LOXN2 re- script localization, though staining signal dropped more
1052 Plant Physiol. Vol. 141, 2006

9. A New Pea Lipoxygenase Involved in Abiotic and Biotic Stress
in resistant than in susceptible tissues. These findings full-length cDNA of LOXN2 (EMBL accession no. AJ749702) was obtained by
5#- and 3#-RACE approaches. The combination of primer 1FW (5#-CCTGAA-
seem to confirm the occurrence of a different degree or TACGAAGAGCTCGCTAAGG-3#) and an oligo(dT) allowed amplicons con-
mechanism of transcriptional regulation between the taining the 3# end of 530 bp to be obtained. In 5#-RACE, the manufacturer’s
two genotypes. The difference in stain distribution be- instructions were followed (Invitrogen). The RNA (1 mg) was reverse tran-
tween uninfected and infected roots suggests that the scribed using primer 4BW; the 5# modified single-strand cDNA was amplified
LOXN2 might work in concert with other enzymes (e.g. using the LOXN2 nested reverse primer 5BW (5#-CATTTTCATTACTGTTT-
GAAG-3#) and the adapter oligonucleotide provided in the kit as forward
anionic peroxidases) in limiting syncytium develop- primer. A product of 267 bp was achieved, which harbored the 5#-UTR.
ment by strengthening the cell walls close to nematode To search for introns, a set of PCR experiments was performed on genomic
feeding sites (Ros Barcelo et al., 1989; Zacheo et al., 1997). DNA (100 ng) of Progress 9 and MG103738. The primer combinations covered
Likewise, LOX action leads to membrane degradation the full-length LOXN2 cDNA and were 6FW (5#-ATGACTCCATTACT-
GAAAGG-3#)/6BW (5#-TCAGTTTTGTACTTTTCCACG-3#), 5FW (5#-CCAAA-
and related cell death in oxidative conditions (Melillo ACATGGAAACTTAAAC-3#)/4BW, 3FW/3BW, and 2FW/1BW (5#-ACA-
et al., 1990) as a result of the synthesis of jasmonic acid, GACATAATAGTTTTTTATTTTCAG-3#). The sequences of products from
known to be involved in senescence, necrosis, and HR both genomic DNA and cDNAs were aligned, and seven introns were located
(Creelman and Mullet, 1997; Rusterucci et al., 1999). in LOXN2 (EMBL accession no. AJ749704). All PCR products of interest were
Indeed, there is evidence that LOXs play an important cloned into pGEM-T Easy Vector (Promega) and propagated in Escherichia coli
`
XL1 blue cells. Sequencing was performed by the CRIBI service (Universita di
role in plant-pathogen interactions by initiating mem- Padova, Italy).
brane damage during the HR through either direct or
indirect promotion of lipid peroxidation (Brash, 1999;
Maccarone et al., 2000). Our data provide evidence that a Southern Blot
newly identified LOX gene in pea is regulated by Genomic DNA of both genotypes was extracted from 1 g of pea roots
wounding, and, additionally, they suggest that LOXN2 (Dellaporta et al., 1983), and 8 mg were digested with EcoRI, HindIII, and SacI,
expression in infected roots, at the level of injured cells fractionated in a 0.8% (w/v) agarose gel, and transferred onto a nylon
membrane (Hybond N1; Amersham Biosciences UK) following standardized
and induced syncytia, seems to be a direct response to procedures (Sambrook et al., 1989). A 1.1-kb LOXN2-specific probe from exon
signals from the nematode. 2 to exon 6 (bases 968–2,045, referring to AJ749704) was chosen and labeled
with DIG, using a PCR-DIG probe synthesis system (Roche Diagnostics
GmbH). The genomic fragment was amplified using the couple of primers
MATERIALS AND METHODS 5FW (5#-CCAAAACATGGAAACTTAAAC-3#) and 4BW (5#-CCATAGAGTT-
GCCTATCTACC-3#). High-stringency hybridization was performed at 42°C
Plant Material, Nematodes, and Treatments in DIG Easy Hyb solution (Roche Diagnostics GmbH) overnight, and the filter
was washed twice at room temperature in 2 3 SSC/0.1% (w/v) SDS for 5 min,
All experiments were performed using a germplasm from pea (Pisum followed by two washes for 15 min at 65°C in 0.5 3 SSC/0.1% (w/v) SDS. The
sativum subsp. transcaucasicum Govorov; Gatersleben collection; accession no. signals were detected by alkaline phosphatase-conjugated anti-DIG antibody
MG103738) and a commercial cultivar Progress 9, resistant and susceptible to and CDP Star (Roche Diagnostics GmbH) chemiluminescent substrate reac-
the cyst nematode Heterodera goettingiana, respectively. Seeds of both geno- tion. The membrane was exposed to high performance chemiluminescence
types were surface sterilized, germinated on filter paper, transferred in clay films (Amersham Biosciences UK) at room temperature for 30 min.
pots containing 10 mL of sterilized sand, and maintained in a growth chamber
with a light intensity of 200 mmol m22 s21 at 19°C with a 16-/8-h light/dark
cycle. Ten-day-old seedlings were inoculated with batches of 50 freshly Northern Blot and RT-PCR Analysis
hatched sterile second-stage juveniles of H. goettingiana, obtained from cysts
collected from a culture maintained on pea. Pools of 0.5-cm infected roots Total RNAwas extracted according to the manufacturer’s instructions (Trizol;
were sampled at time intervals of 24 and 48 h following nematode infection, Invitrogen) and used in northern and RT-PCR analyses. RNA (5 mg/lane) was
immediately frozen in liquid nitrogen, and stored at 270°C until required. separated on a 1.2% (w/v) agarose-formaldehyde gel and transferred to Hybond
Noninfected roots were used as controls. N1 membrane (Amersham Biosciences UK). Hybridization was carried out
For wounding experiments, seeds of both pea genotypes were surface overnight at 42°C with Ultrahyb buffer (Ambion) containing formamide,
sterilized, germinated on filter paper, soaked in tap water, transferred in followed by two washes in 2 3 -1 3 SSC/0.1% (w/v) SDS at 60°C for 10 min
Hoagland solution, and maintained in growth chambers at the same condi- and one wash in 0.1 3 SSC/0.1% (w/v) SDS. Filters were exposed to Kodak
tions as reported above. The roots of 1-week-old seedlings were injured by a BIOMAX films (Amersham Biosciences UK) for at least 4 h. A LOXN2-specific
needle at three different points (from the apices to elongation zones). Punc- probe spanned the 3#-UTR region (bases 2,475–2,650, referring to AJ749702),
tured seedlings were laid on filter paper and the roots covered with filter whereas the probe to detect the abundance of other LOX members included the
paper soaked in Hoagland solution. Samplings (1 cm) of injured roots and conserved ORF region (bases 994–2,160, referring to AJ749702). The probes were
controls were collected at 3, 6, 12, and 24 h after wounding. radiolabeled with 32dCTP using the Ready Primer kit (Amersham Biosciences
UK). The optical density (OD) of the signal bands was determined by the ID
Image Analysis software (Kodak Digital Science), and the relative OD was
Cloning of Full-Length LOXN2 cDNA graphed as histograms (Microsoft Excel) representing the ratio between OD of
and Genomic Fragments LOX genes and 26S rRNA (checking for the equal loading of RNA).
Semiquantitative RT-PCR was performed on single-strand cDNAs derived
A PCR-based strategy was used to isolate LOX cDNA fragments from pea from DNase-treated RNA (2 mg) and reverse transcribed (AMV) by oligo(dT)
roots using degenerate primers, which were designed from conserved func- primers, according to the provider (Roche Diagnostics). The PCR reaction was
tional domains of plant LOX sequences. Total RNA (2 mg) of resistant and conducted in 50 mL total volume containing cDNA (2 mL), 5 mM MgCl2, 200 mM
susceptible genotypes was reverse transcribed using an oligo(dT) and single- of each dNTP, 10 pmol of each specific primer, and one unit of Taq DNA
strand cDNA used in PCR reactions (see details in the RT-PCR paragraph). The polymerase (Roche Diagnostics). The cycle parameters were as follows:
primer combinations 2FW (5#-KGARCCATTCATCATAGCAAC-3#)/2BW denaturation at 94°C for 2 min; 30 cycles at 94°C 30 s, 60°C 30 s, and 72°C
(5#-GAGCTCTTCGTATTCAGGAGA-3#), 3FW (5#-GCATGGATGACYGATG- 30 s; and final extension at 72°C for 7 min. LOXN2 message was amplified by
ARGAA-3#)/3BW (5#-ATCAAAGTATTTCGCGCACCG-3#), and 4FW (5#-RRG- primer 1FW (5#-CCTGAATACGAAGAGCTCGCTAAGG-3#) and an anchor
GSACWKTRGTGTTGATGC-3#)/4BW (5#-CCATAGAGTTGCCTATCTACC-3#; primer (5#-GACCACGCGTATCGATGTCGAC-3#), and the product was 530 bp.
K 5 G, T; R 5 A, G; Y 5 C, T; S 5 C, G; W 5 A, T) produced single amplicons of The 26S rRNA fragment (approximately 500 bp) was amplified by primers
658, 534, and 1,072 bp, respectively, and the sequencing of cloned fragments 26SFW (5#-AGCATTGCGATGGTCCCTGCGG-3#) and 26SBW (5#-GCCCCG-
revealed that amplified products shared homology with LOX sequences. The TCGATTCAGCCAAACTCC-3#), and the signal was used to check for
Plant Physiol. Vol. 141, 2006 1053

10. Veronico et al.
equal amounts of cDNA template. A mock reaction was also performed to sense primer (5#-TCATCCTAGGATGACTCCATTACTGAAA-3#) and the an-
check for DNA contamination in RNA samples. Diagrams were designed by tisense primer (5#-ATAAGAATGCGGCCGCTTAGATAGAGATACTGTT-3#),
measuring band signal intensity normalized with respect to 26S signals. respectively. This LOXN2 was cloned in the AvrII-NotI-oriented direction into
Northern-blot and RT-PCR experiments were carried out several times with the expression vector pPIC9 for Pichia pastoris (Invitrogen). The recombinant
independent RNA extracts. Standard errors were calculated and indicated as plasmids harboring the yeast a-factor signal in-frame with LOXN2 coding
size bars. All data sets were subjected to the Student’s t test, and those sequence were selected by PCR control and sequence analysis. For yeast
presented in this work have a P , 0.05. transformation, 10 mg of SalI-linearized recombinant vector was transferred
into GS115 Pichia host strain using a GenePulser electroporator (Bio-Rad
Laboratories) and subsequently plated on minimal methanol agar substrate.
In Situ Hybridization Negative controls included clones containing empty, unmodified vectors.
The roots were collected 48 h after nematode infection and fixation (4% [w/v] The recombinant colonies were first grown in 1 mL of buffered glycerol
formaldehyde in phosphate-buffered saline), embedding in paraffin, and in (0.1 M potassium phosphate, pH 6.0, 13.4 g/L of yeast nitrogen base without
situ hybridization carried out as described by Jackson (1991). The LOXN2- amino acids, 400 mg/L biotin, and 1% [v/v] glycerol). The cells (1 OD600) were
specific probe spanned the 3#-UTR region from nt 2,475 to nt 2,650 (with recovered by centrifugation, suspended in 1 mL BMM (0.1 M potassium
reference to AJ749702). Sense and antisense DIG-labeled riboprobes were phosphate, pH 6.0, 13.4 g/L of yeast nitrogen base without amino acids,
produced by in vitro transcription of linearized plasmid DNA (cut with either 400 mg/L biotin, and 0.5% [v/v] methanol), and grown in 15-mL tubes at 30°C.
SpeI or NcoI) using T7 and SP6 RNA polymerase, respectively (Roche Diag- Methanol (0.5% [v/v]) was added daily. After 3 d the cells were precipitated
nostics). Hybridization was performed at 54°C overnight using 50 ng of by centrifugation at 13,000g and the supernatant used for western-blot
riboprobe. The slides were soaked twice in prewarmed (54°C) 0.2 3 SSC for 30 analysis. For large-scale biosynthesis, one Pichia transformant was grown in
min, rinsed twice for 5 min with NTE solution (500 mM NaCl, 10 mM Tris-HCl, 50 mL of buffered minimal glycerol to generate sufficient biomass, and the
pH 8, 1 mM EDTA) at 37°C, and incubated at the same temperature for 30 min cells recovered by centrifugation and suspended in 0.2 L of BMM.
in prewarmed NTE solution containing RNase A (20 mg/mL). The slides were LOXN2 biosynthesis was induced by adding 0.5% (v/v) methanol in BMM
then rinsed twice for 5 min in NTE solution at 37°C, and washed 1 h in 0.2 3 for 3 d. The cells were then precipitated by centrifugation, and ammonium
SSC at 54°C and 5 min in phosphate-buffered saline at room temperature. The sulfate up to 70% was added to the supernatant. The precipitated proteins
signals were detected using an alkaline phosphatase-conjugated antibody were centrifuged at 10,000g at 4°C for 30 min, resuspended in 5 mL of 0.1 M
(1:1,000), and the reaction was stopped by adding Tris-EDTA. Sections were sodium phosphate buffer, pH 6.5, and dialyzed against 4 L of 0.1 M sodium
mounted in Aquamount (BDH Laboratory Supplies) on glass slides and phosphate buffer, pH 6.5, overnight at 4°C. An aliquot of proteins was assayed
viewed under a bright-field microscope. Controls to check for signal back- for LOX activity.
ground were: A, samples hybridized with LOXN2 probe in sense orientation;
and B, untreated samples reacting with alkaline phosphatase. Western Blot
Total proteins, extracted from recombinant Pichia for LOXN2, were sepa-
Sequence Analyses, Alignment, and Phylogenetics
rated by SDS-PAGE (12% [w/v] acrylamide). Samples were heated at 95°C for
LOXN2 full-length cDNA and its deduced protein were first aligned with 5 min prior to loading. The gel was calibrated for molecular mass with prestained
other LOX sequences by means of ClustalW (http://www.ebi.ac.uk.clustalw). MultiMark multicolored standard (Invitrogen). After electrophoresis, proteins
ClustalW and visual inspection (PILEUP program) were used to optimize and were transferred onto nitrocellulose membrane by semidry blotting according to
produce the final alignments. Bio-Rad Laboratories’ manual instruction. Blots were probed with pea anti-LOX
Phylogenetic trees based on either nucleotide or amino acid sequences and polyclonal antibodies (Domoney et al., 1990), diluted 1:5,000 in blocking solution
reconstructed using different approaches were obtained using PAUP 4.0 b10 (BLOTTO: 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% [v/v] Triton X-100,
package (Swofford, 1998), and bootstrap values were based on 1,000 repli- containing 5% [w/v] nonfat dry milk) overnight at 4°C. The membrane was
cates. Bootstrap values assess the degree of support for each branch on the washed with the same blocking solution and incubated for 1 h with an anti-rabbit
trees, and the 80% value was accepted as an indicative of a well-supported IgG conjugated to horseradish peroxidase diluted 1:5,000 in blocking solution.
branch. The sequences of the monocot plants (barley [Hordeum vulgare], maize Blots were revealed for peroxidase activity by enhanced chemiluminescence
[Zea mays], and rice [Oryza sativa]) were used as an outgroup in the phylo- (ECL Plus kit; Amersham Biosciences UK).
genetic reconstructions.
LOXN2 Activity
Molecular Modeling
One unit of LOX activity is the amount of enzyme required to produce 1 mmol
The pea LOXN2 protein structure was modeled using Swissmodel set of HPO and hydroxy acid per minute at 25°C (Hughes et al., 1998). LOXN2
automatically to seek appropriate known protein crystal structures as tem- activity was determined spectrophotometrically by monitoring the increase in
plates based on sequence similarity. The selected five template structures A234 resulting from the formation of conjugated diene structures from exoge-
(protein structure data bank accession codes 1RRL, 1JNQ, 1N8Q, 1RRH, and nously supplied polyunsaturated fatty acids as substrates. To determine the
1RRL) were all from another legume, soybean (Glycine max) LOX (LOX3), all optimum pH of the reaction, the assays of LOX activity were carried out with
˚
solved at 2.1 A resolution or better, and two of which were solved with ligands linoleic, linolenic, and arachidonic acids (0.3 mM) testing the following buffers for
(epigallocathechin or protocatechuic acid) in the active site adjacent to the each substrate: 0.1 M sodium acetate buffer, pH 4.5; 0.1 M sodium phosphate
bound iron ion, and energy minimalization was carried out with the buffer, pH 5.5, 6.0, and 6.5; and 100 mM Tris-HCl, pH 8.0, at 25°C.
GROMOS96 program. When this was repeated but with the program set to
use a new refinement of the structure of soybean LOX1 (PDB accession no.
1F8N) as the template, an improved model was obtained, although no
Analysis of LOXN2 Products
template is available with a bound ligand or substrate. The geometry of the Crude extracts in 0.1 M sodium phosphate buffer, pH 6.5, from recombinant
model was satisfactory as checked online with the PROCHECK suite of and control yeasts were incubated for 30 min in 1 mL of 0.1 M sodium
programs. The Ramachandran plot showed 88.2% of amino acid positions phosphate buffer, pH 6.0, or 0.1 M sodium acetate buffer, pH 4.5, containing
lying in the most favored regions, 11.2% in additional allowed regions, and 0.3 mM linoleic acid. Reaction products were reduced with sodium borohy-
only 0.4% in generously allowed regions. Only 0.2% of the residues were in dride, extracted with chloroform/methanol (2:1, v/v), and dried. The reaction
normally disallowed regions, but most of these are Glys and present in a small products were resuspended in methanol/water/acetic acid (85:15:0.1, v/v/v)
region close to the N terminus or in loop regions. The coordinates of the model and separated by reverse-phase HPLC using a C18 Ultrasphere column
are available by e-mail from the corresponding author. (Beckmann; 0.46 3 25 cm) as described previously (Santino et al., 2003). The
peak adsorbing at 234 nm containing the HODE was collected, dried, and
Synthesis of LOXN2 in Yeast resuspended in n-hexane/propan-2-ol/acetic acid (100:2:0.1, v/v/v). The
9-HODE and 13-HODE were separated by straight-phase HPLC with a Silica
The ends of the LOXN2 coding region (2.6 kb) were modified to include an Ultrasphere column (Phenomenex; 250 3 4.60 mm, 5 mm). Authentic stan-
AvrII site upstream the ATG start codon and a NotI site at the 3# end using the dards of 9- and 13-HODE were purchased from ICN. HPLC analyses were
1054 Plant Physiol. Vol. 141, 2006

11. A New Pea Lipoxygenase Involved in Abiotic and Biotic Stress
carried out using a Beckman System Gold apparatus (Beckman Coulter) May C, Hohne M, Gnau P, Schwennesen K, Kindl H (2000) The N-terminal
equipped with a 126 solvent module, a 168 detector, and an Applera Series beta-barrel structure of lipid body lipoxygenase mediates its binding to
apparatus (Applera Europe B.V.). liposome and lipid bodies. Eur J Biochem 267: 1100–1109
McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two en-
Sequence data from this article can be found in the GenBank/EMBL data dogenous proteins that induce cell wall extension in plants. Plant Cell 4:
libraries under accession numbers AJ769702 and AJ769704. 1425–1433
Melillo MT, Bleve-Zacheo T, Zacheo G, Perrino P (1990) Morphology and
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ACKNOWLEDGMENTS goettingiana. Nematol Medit 18: 83–91
The excellent assistance with figures from Dr. Alberto Troccoli is gratefully Minor W, Steczko J, Stec B, Otwinowski Z, Bolin JT, Walter R, Axelrod B
˚
(1996) Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.
acknowledged. We thank Mr. Roberto Lerario for his technical assistance.
Biochemistry 35: 10687–10701
Received April 7, 2006; revised April 7, 2006; accepted April 26, 2006; Moy P, Qutob D, Chapman BP, Atkinson I, Gijzen M (2004) Patterns of
published May 5, 2006. gene expression upon infection of soybean plants by Phytophthora sojae.
Mol Plant Microbe Interact 17: 1051–1062
Pilet-Nayel L, Muehlbauer FJ, McGee RJ, Kraft JM, Baranger A, Coyne CJ
(2002) Quantitative trait loci for partial resistance to Aphanomyces root
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