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First published online May 5, 2006; 10.1104/pp.106.081679 Plant Physiology 141:1045-1055 (2006) © 2006 American Society of Plant Biologists A Novel Lipoxygenase in Pea Roots. Its Function in Wounding and Biotic StressInstitute 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, E.C. 1.13.11.12) are nonheme iron-containing enzymes that catalyze the addition of molecular oxygen at either the C-9 or C-13 residue of fatty acids with a 1,4-pentadiene structure. Linoleic and linolenic acids are the most abundant fatty acids in the lipid fraction of plant membranes and are the major substrates for LOXs. The oxygenation step leads to a reaction cascade (termed the LOX pathway), in which the hydroperoxides (HPOs), produced by the LOX activity, are substrates of HPO lyases and allene oxide synthases (Vick, 1993  ). HPOs are highly reactive and rapidly degrade into metabolites that are precursors for jasmonic acid, methyl jasmonate, conjugated dienoic acids, and volatile aldehydes. These products are known to play a role in plant defense (Wasternack et al., 1998).
LOXs are encoded by gene families (LOX) in most, if not all, of the plant species studied so far (Royo et al., 1996
Phytoparasitic nematodes are the most widespread and deleterious pests for many crops. Several complex mechanisms, which underlie the interaction between the plant and endoparasitic pathogen, induce the differentiation of host cells into feeding structures necessary for parasite development. In particular, cyst nematodes enter the root and migrate to the vascular cylinder where they select a cell that becomes the initial feeding site. Once the parasite has established, this cell is induced to develop into a syncytium as the result of cell-wall breakdown and subsequent fusion of neighboring cell protoplasm. A particularly serious pest of pea is the cyst nematode Heterodera goettingiana, which is difficult to control due to its long soil persistence and resistance to chemical control (Thompson et al., 2000
In the past decade, a considerable number of pea genes responsible for resistance against a range of pathogens, but not nematodes, have been characterized (Chang et al., 1995 This study focused on the molecular cloning and characterization of LOXN2, a member of the pea LOX family genes, during plant-nematode interaction. LOXN2 represented a novel isoform in the pea able to produce both C-9 and C-13 HPOs. The gene transcription was down-regulated upon nematode infection in the resistant genotype, exhibiting an inverse trend to that of other LOX family members. Moreover, the message localization in the resistant and susceptible genotypes leads us to hypothesize a role for LOXN2 in the antinematode defense system.
The LOXN2 cDNA, Encoded Protein, and Protein Model Structure
The full-length cDNA of LOXN2 (EMBL accession no. AJ749702) harbored an open reading frame (ORF) of 2,481 nt encoding a presumptive translation product of 826 amino acids with a Mr of 93,787 and a pI of 5.18 (ProtParam tool: Wilkins et al., 1998
The predicted protein product LOXN2 showed 57.4%, 57.1%, and 58.2% identity and 84.4%, 85.6%, and 84.9% similarity to pea LOXN1 (EMBL accession no. AAB71759), LOXN4 (EMBL accession no. CAC04380), and LOXN5 (EMBL accession no. CAA75609), respectively. LOXN2 contained all the functional domains typical of LOXs (Fig. 1
): an N-terminal region forming a
A stretch of approximately 50 amino acids (positions 1–52) in the N-terminal region differed from those of other pea LOXs. However, a high degree of similarity was observed in the following region, spanning over 100 residues, which is predicted to be a  -barrel structure by standard secondary structure analysis programs (e.g. National Center for Biotechnology Information conserved domain search) and three-dimensional molecular modeling (see below). In the C-terminal region, LOXN2 has two deletions of six and 27 amino acids as compared to the stretches 277 to 281 and 296 to 322 of the soybean (Glycine max) LOX1 (Swiss-Prot accession no. P08170), which is one of the best characterized among LOXs. A molecular model of LOXN2 was produced by homology modeling using five templates for crystal structures of LOX proteins from another legume, soybean (Fig. 2
).
The deletions located in the domain III (Fig. 1) lead to the absence of an external loop, but did not change the position and conformation of the -barrel and the catalytic site of LOXN2 (Fig. 2). The residues involved in iron binding (His-486, His-491, His-677, Asn-688, and Ile-826) were conserved in domain V. The positional specificity of plant LOXs is usually determined by the motifs R/TH or R/TF for 13-LOX and R/TV for 9-LOX in the active site (Feussner and Wasternack, 2002). Unusually, LOXN2 contained a Thr-Tyr motif (R/TY; Fig. 1), which has not been described previously to our knowledge. A phylogenetic tree (Fig. 3 ) was constructed based on gap-free multi-alignments of dicot and monocot LOX sequences currently available. Pea LOXN2 fell into the highly supported monophyletic group of leguminous species. However, its closest relative was not the other previously identified pea LOX (LOXN5) but lentil (Lens culinaris) LOX (0.22 substitutions/site). Indeed, within leguminous species, two well-supported clusters were found, each containing a different pea LOX sequence. Interestingly, none of the closely related LOX showed the deletions described for the pea LOXN2.
Biochemical Activity of Recombinant Yeast LOXN2 To study the biochemical function and to test whether the lack of a loop in the C terminus affected its activity, LOXN2 was cloned in the expression vector pPIC9 and over-expressed in Pichia pastoris (strain GS115). In induced strains (see "Materials and Methods"), LOXN2 was represented by a strong band of expected size (approximately 94 kD) in both silver-stained polyacrylamide gels and western blots, whereas no signal was revealed in control strains harboring the empty vector (Fig. 4 ).
The biochemical activity of recombinant LOXN2 was investigated in extracts of yeast expressing the protein (Table I ) by measuring the increase of A234 using linoleic, linolenic, and arachidonic acids as substrates. The optimum pH was determined for each substrate by varying the pH of the reaction buffers. The activity of LOXN2 was detected with all the substrates in a range of pH 4.5 to 8.0. In particular, at pH 4.5, the maximum activity of 1.35 units/mg protein occurred with linoleic acid, whereas it fell to 0.23 units/mg protein and 0.18 units/mg protein with linolenic and arachidonic acids, respectively. However, at pH 6.0, the maximum activity was 0.45 units/mg protein with arachidonic acid, followed by 0.18 units/mg protein and 0.07 units/mg protein with linolenic and linoleic acids, respectively. The extracts from controls did not show any activity.
Plant LOXs are usually classified as 9-LOXs and 13-LOXs on the basis of their product specificities. As pea LOXN2 was found to contain the unusual R/TY motif in the active site, its positional specificity was tested. Linoleic acid was selected as substrate in 0.1 M sodium acetate buffer at pH 4.5 and 0.1 M sodium phosphate buffer at pH 6.0. The reaction products were reduced with sodium borohydride and separated by reverse-phase HPLC. The peak adsorbing at 234 nm and containing the HPOs was collected and subjected to straight-phase HPLC to separate 13- and 9-hydroxy octadecadienoic acid isomer (HODE). The retention times of LOXN2 reaction products were consistent with both authentic standards of 9- and 13-HODE and those of soybean LOX1. In the presence of both buffers, LOXN2 produced 9- and 13-HODE in a 3:1 ratio (Table I).
Genomic DNA of MG103738 and Progress 9 was endonuclease restricted, size fractionated, transferred onto a nylon membrane, and hybridized with a digoxigenin (DIG)-labeled genomic DNA probe spanning 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 genotypes. The sizes of DNA fragments detected were consistent with those predicted from the restriction map of genomic sequences. These results suggest the occurrence of one LOXN2 copy and a very high degree of identity in both genotypes.
To search for introns, PCR experiments were performed on genomic DNA with primer combinations designed along the full-length transcript. The sequence comparison confirmed that LOXN2 was identical in both genotypes and contained eight exons and seven introns (Fig. 5A). The exon positions were conserved with respect to pea LOXN5 (GenBank accession no. Y15410), in which the exons 6 and 7 were separated by an intron that is absent in LOXN2 (Fig. 5A). Moreover, LOXN2 introns were rich in A/T nucleotides (73%) and harbored the canonical splicing GT/AG motifs.
Pea LOXs represent a gene family with a discrete polymorphism in the 3' ends among the members. Therefore, a specific probe was designed in the 3'-UTR (Fig. 5A) to monitor LOXN2 expression in roots under abiotic and biotic stresses. Because nematodes invade host roots by cell perforation using their stylet, changes in LOXN2 gene transcription following mechanical injuries were examined. The root elongation zone and apices of 1-week-old MG103738 and Progress 9 seedlings were needle punctured, collected at different time intervals, and LOXN2 transcript detected by semiquantitative reverse transcription (RT)-PCR (Fig. 6, A and B ). In unwounded plants, the variation of LOXN2 expression appeared to be constant and the message abundance 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 decrease phase, LOXN2 transcript abundance in MG103738 was 10 to 8 times higher than in the control, while in Progress 9 it fell down to the level of its control. LOXN2 message levels in MG103738 at 24 h were 16-fold higher than in the control, whereas the message abundance in Progress 9 was 8 times higher than in the unwounded control samples. Taken together, these data suggest that LOXN2 maintains a higher grade of expression in MG103738 than in Progress 9.
Global LOX Transcription Is Triggered in the Infected Resistant Genotype, But LOXN2 Is Repressed Transcript abundance was monitored in nematode-challenged roots of MG103738 and Progress 9 genotypes and compared to uninfected root tissues. Northern analyses were performed at 24 and 48 h after H. goettingiana infection using two distinct probes (Fig. 5; see also "Materials and Methods"): one contained a highly conserved stretch in the ORF and was used to detect a message pool from LOX members (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 susceptible 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 contrary, LOXN2 transcript (Fig. 7B), 24 and 48 h following nematode infection, was down-regulated in both infected resistant (0.6- and 0.8-fold versus control) and susceptible roots (0.79- and 0.8-fold versus control).
Localization of LOXN2 Message upon Nematode Infection To determine the spatial expression pattern of LOXN2 during nematode infection, in situ experiments were conducted on serial cross sections of uninfected and 48 h infected roots. Sense and antisense riboprobes spanning the LOXN2 3'-UTR were used (Fig. 5A). In uninfected roots, the transcript was visualized in cortical and endodermal cells as a purple staining, which appeared more intense in the resistant than in the susceptible genotype (Fig. 8, A and B ). In resistant infected roots, faint and infrequent signal spots were observed in the cortex, but LOXN2 message was abundant in the outer cells surrounding the vascular cylinder, in those cells injured by nematode penetration and those flanking the induced syncytia (Fig. 8, C and E). In susceptible infected roots, the transcript was detected in cortical cells damaged by nematode invasion and undergoing necrosis, and in parenchymatous cells of the vascular cylinder strictly related to syncytia development (Fig. 8D). In MG103738 roots, syncytia rapidly degenerated and were highly reactive to the probe due to the condensed cytoplasm, which is a feature of collapse (Fig. 8, E and D). No LOXN2 mRNA was detected in infected and uninfected root sections when hybridized with a sense probe (Fig. 8, G and H).
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 report, we focused on the structure, the transcription pattern, and localization of LOXN2 in response to the cyst nematode H. goettingiana and the biochemical properties of the enzyme. The unusual structural features of LOXN2 are, surprisingly, not reflected in an alteration of the biochemical activity of the enzyme.
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
Plant LOXs are classified into 9- and 13-LOXs according to the position at which the oxygenation of linoleic acid occurs. Most of the plant LOXs harbors a tightly conserved Arg in the substrate-binding pocket. In 13-LOXs of several plants, the occurrence of His or Phe has been proposed to mask the positive charge of Arg and to hamper the interaction with the linoleate carboxylate. This should favor the entry of the linoleate methyl terminus and the C-13 oxidation (Hornung et al., 1999
Accumulation of LOXN2 transcripts over their normal basal level was induced by wounding in both resistant and susceptible genotypes. The LOXN2 response to wounding was remarkably rapid; maximum transcript levels were reached within 3 h after treatment and they were transient and biphasic. The resistant genotype reacted more strongly than the susceptible genotype in increasing LOXN2 expression to wounding. A similar kinetic expression of LOX with DPS has been reported in maize (Zea mays) in response to exogenous methyl jasmonic acid treatment (Kim et al., 2003
Following nematode infection, the abundance of mRNA representing root LOXs increased in the resistant (but not in the susceptible) genotype while mRNA of LOXN2 decreased, indicating that LOX family members responded differentially to the infection. Triggering of plant LOX gene transcription is the most frequently observed response to pathogen attacks (Porta and Rocha-Sosa, 2002
In uninfected peas, the occurrence of LOXN2 transcript in cortical and endodermal cells suggests a central function for this enzyme in root development. In light of these results, it can be hypothesized that LOXN2 with acidic pH optimum, as for other proteins like expansins, could mediate relaxation of cell walls during acid-induced growth (McQueen-Mason et al., 1992
Plant Material, Nematodes, and Treatments All experiments were performed using a germplasm from pea (Pisum sativum subsp. transcaucasicum Govorov; Gatersleben collection; accession no. MG103738) and a commercial cultivar Progress 9, resistant and susceptible to the cyst nematode Heterodera goettingiana, respectively. Seeds of both genotypes were surface sterilized, germinated on filter paper, transferred in clay pots containing 10 mL of sterilized sand, and maintained in a growth chamber with a light intensity of 200 µmol m–2 s–1 at 19°C with a 16-/8-h light/dark cycle. Ten-day-old seedlings were inoculated with batches of 50 freshly 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 were sampled at time intervals of 24 and 48 h following nematode infection, immediately frozen in liquid nitrogen, and stored at –70°C until required. Noninfected roots were used as controls. For wounding experiments, seeds of both pea genotypes were surface sterilized, germinated on filter paper, soaked in tap water, transferred in Hoagland solution, and maintained in growth chambers at the same conditions as reported above. The roots of 1-week-old seedlings were injured by a needle at three different points (from the apices to elongation zones). Punctured seedlings were laid on filter paper and the roots covered with filter paper soaked in Hoagland solution. Samplings (1 cm) of injured roots and controls were collected at 3, 6, 12, and 24 h after wounding.
A PCR-based strategy was used to isolate LOX cDNA fragments from pea roots using degenerate primers, which were designed from conserved functional domains of plant LOX sequences. Total RNA (2 µg) of resistant and susceptible genotypes was reverse transcribed using an oligo(dT) and single-strand cDNA used in PCR reactions (see details in the RT-PCR paragraph). The primer combinations 2FW (5'-KGARCCATTCATCATAGCAAC-3')/2BW (5'-GAGCTCTTCGTATTCAGGAGA-3'), 3FW (5'-GCATGGATGACYGATGARGAA-3')/3BW (5'-ATCAAAGTATTTCGCGCACCG-3'), and 4FW (5'-RRGGSACWKTRGTGTTGATGC-3')/4BW (5'-CCATAGAGTTGCCTATCTACC-3'; K = G, T; R = A, G; Y = C, T; S = C, G; W = A, T) produced single amplicons of 658, 534, and 1,072 bp, respectively, and the sequencing of cloned fragments revealed that amplified products shared homology with LOX sequences. The full-length cDNA of LOXN2 (EMBL accession no. AJ749702) was obtained by 5'- and 3'-RACE approaches. The combination of primer 1FW (5'-CCTGAATACGAAGAGCTCGCTAAGG-3') and an oligo(dT) allowed amplicons containing the 3' end of 530 bp to be obtained. In 5'-RACE, the manufacturer's instructions were followed (Invitrogen). The RNA (1 µg) was reverse transcribed using primer 4BW; the 5' modified single-strand cDNA was amplified using the LOXN2 nested reverse primer 5BW (5'-CATTTTCATTACTGTTTGAAG-3') and the adapter oligonucleotide provided in the kit as forward primer. A product of 267 bp was achieved, which harbored the 5'-UTR. To search for introns, a set of PCR experiments was performed on genomic DNA (100 ng) of Progress 9 and MG103738. The primer combinations covered the full-length LOXN2 cDNA and were 6FW (5'-ATGACTCCATTACTGAAAGG-3')/6BW (5'-TCAGTTTTGTACTTTTCCACG-3'), 5FW (5'-CCAAAACATGGAAACTTAAAC-3')/4BW, 3FW/3BW, and 2FW/1BW (5'-ACAGACATAATAGTTTTTTATTTTCAG-3'). The sequences of products from both genomic DNA and cDNAs were aligned, and seven introns were located in LOXN2 (EMBL accession no. AJ749704). All PCR products of interest were cloned into pGEM-T Easy Vector (Promega) and propagated in Escherichia coli XL1 blue cells. Sequencing was performed by the CRIBI service (Università di Padova, Italy).
Genomic DNA of both genotypes was extracted from 1 g of pea roots (Dellaporta et al., 1983
Total RNA was extracted according to the manufacturer's instructions (Trizol; Invitrogen) and used in northern and RT-PCR analyses. RNA (5 µg/lane) was separated on a 1.2% (w/v) agarose-formaldehyde gel and transferred to Hybond N+ membrane (Amersham Biosciences UK). Hybridization was carried out overnight at 42°C with Ultrahyb buffer (Ambion) containing formamide, followed by two washes in 2x-1x SSC/0.1% (w/v) SDS at 60°C for 10 min and one wash in 0.1x SSC/0.1% (w/v) SDS. Filters were exposed to Kodak BIOMAX films (Amersham Biosciences UK) for at least 4 h. A LOXN2-specific probe spanned the 3'-UTR region (bases 2,475–2,650, referring to AJ749702), whereas the probe to detect the abundance of other LOX members included the conserved ORF region (bases 994–2,160, referring to AJ749702). The probes were 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 graphed as histograms (Microsoft Excel) representing the ratio between OD of LOX genes and 26S rRNA (checking for the equal loading of RNA). Semiquantitative RT-PCR was performed on single-strand cDNAs derived from DNase-treated RNA (2 µg) and reverse transcribed (AMV) by oligo(dT) primers, according to the provider (Roche Diagnostics). The PCR reaction was conducted in 50 µL total volume containing cDNA (2 µL), 5 mM MgCl2, 200 µM of each dNTP, 10 pmol of each specific primer, and one unit of Taq DNA polymerase (Roche Diagnostics). The cycle parameters were as follows: denaturation at 94°C for 2 min; 30 cycles at 94°C 30 s, 60°C 30 s, and 72°C 30 s; and final extension at 72°C for 7 min. LOXN2 message was amplified by primer 1FW (5'-CCTGAATACGAAGAGCTCGCTAAGG-3') and an anchor primer (5'-GACCACGCGTATCGATGTCGAC-3'), and the product was 530 bp. The 26S rRNA fragment (approximately 500 bp) was amplified by primers 26SFW (5'-AGCATTGCGATGGTCCCTGCGG-3') and 26SBW (5'-GCCCCGTCGATTCAGCCAAACTCC-3'), and the signal was used to check for equal amounts of cDNA template. A mock reaction was also performed to check for DNA contamination in RNA samples. Diagrams were designed by measuring band signal intensity normalized with respect to 26S signals. Northern-blot and RT-PCR experiments were carried out several times with independent RNA extracts. Standard errors were calculated and indicated as size bars. All data sets were subjected to the Student's t test, and those presented in this work have a P < 0.05.
The roots were collected 48 h after nematode infection and fixation (4% [w/v] formaldehyde in phosphate-buffered saline), embedding in paraffin, and in situ hybridization carried out as described by Jackson (1991)
LOXN2 full-length cDNA and its deduced protein were first aligned with other LOX sequences by means of ClustalW (http://www.ebi.ac.uk.clustalw). ClustalW and visual inspection (PILEUP program) were used to optimize and produce the final alignments.
Phylogenetic trees based on either nucleotide or amino acid sequences and reconstructed using different approaches were obtained using PAUP 4.0 b10 package (Swofford, 1998
The pea LOXN2 protein structure was modeled using Swissmodel set automatically to seek appropriate known protein crystal structures as templates based on sequence similarity. The selected five template structures (protein structure data bank accession codes 1RRL, 1JNQ, 1N8Q, 1RRH, and 1RRL) were all from another legume, soybean (Glycine max) LOX (LOX3), all solved at 2.1 Å resolution or better, and two of which were solved with ligands (epigallocathechin or protocatechuic acid) in the active site adjacent to the bound iron ion, and energy minimalization was carried out with the 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 template is available with a bound ligand or substrate. The geometry of the model was satisfactory as checked online with the PROCHECK suite of programs. The Ramachandran plot showed 88.2% of amino acid positions lying in the most favored regions, 11.2% in additional allowed regions, and only 0.4% in generously allowed regions. Only 0.2% of the residues were in normally disallowed regions, but most of these are Glys and present in a small region close to the N terminus or in loop regions. The coordinates of the model are available by e-mail from the corresponding author.
The ends of the LOXN2 coding region (2.6 kb) were modified to include an AvrII site upstream the ATG start codon and a NotI site at the 3' end using the sense primer (5'-TCATCCTAGGATGACTCCATTACTGAAA-3') and the antisense primer (5'-ATAAGAATGCGGCCGCTTAGATAGAGATACTGTT-3'), respectively. This LOXN2 was cloned in the AvrII-NotI-oriented direction into the expression vector pPIC9 for Pichia pastoris (Invitrogen). The recombinant plasmids harboring the yeast The recombinant colonies were first grown in 1 mL of buffered glycerol (0.1 M potassium phosphate, pH 6.0, 13.4 g/L of yeast nitrogen base without amino acids, 400 µg/L biotin, and 1% [v/v] glycerol). The cells (1 OD600) were recovered by centrifugation, suspended in 1 mL BMM (0.1 M potassium phosphate, pH 6.0, 13.4 g/L of yeast nitrogen base without amino acids, 400 µg/L biotin, and 0.5% [v/v] methanol), and grown in 15-mL tubes at 30°C. Methanol (0.5% [v/v]) was added daily. After 3 d the cells were precipitated by centrifugation at 13,000g and the supernatant used for western-blot analysis. For large-scale biosynthesis, one Pichia transformant was grown in 50 mL of buffered minimal glycerol to generate sufficient biomass, and the cells recovered by centrifugation and suspended in 0.2 L of BMM. LOXN2 biosynthesis was induced by adding 0.5% (v/v) methanol in BMM for 3 d. The cells were then precipitated by centrifugation, and ammonium sulfate up to 70% was added to the supernatant. The precipitated proteins were centrifuged at 10,000g at 4°C for 30 min, resuspended in 5 mL of 0.1 M sodium phosphate buffer, pH 6.5, and dialyzed against 4 L of 0.1 M sodium phosphate buffer, pH 6.5, overnight at 4°C. An aliquot of proteins was assayed for LOX activity.
Total proteins, extracted from recombinant Pichia for LOXN2, were separated by SDS-PAGE (12% [w/v] acrylamide). Samples were heated at 95°C for 5 min prior to loading. The gel was calibrated for molecular mass with prestained MultiMark multicolored standard (Invitrogen). After electrophoresis, proteins were transferred onto nitrocellulose membrane by semidry blotting according to Bio-Rad Laboratories' manual instruction. Blots were probed with pea anti-LOX polyclonal antibodies (Domoney et al., 1990
One unit of LOX activity is the amount of enzyme required to produce 1 µmol of HPO and hydroxy acid per minute at 25°C (Hughes et al., 1998
Crude extracts in 0.1 M sodium phosphate buffer, pH 6.5, from recombinant and control yeasts were incubated for 30 min in 1 mL of 0.1 M sodium phosphate buffer, pH 6.0, or 0.1 M sodium acetate buffer, pH 4.5, containing 0.3 mM linoleic acid. Reaction products were reduced with sodium borohydride, extracted with chloroform/methanol (2:1, v/v), and dried. The reaction products were resuspended in methanol/water/acetic acid (85:15:0.1, v/v/v) and separated by reverse-phase HPLC using a C18 Ultrasphere column (Beckmann; 0.46 x 25 cm) as described previously (Santino et al., 2003 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AJ769702 and AJ769704.
The excellent assistance with figures from Dr. Alberto Troccoli is gratefully acknowledged. We thank Mr. Roberto Lerario for his technical assistance. Received April 7, 2006; returned for revision April 7, 2006; accepted April 26, 2006.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Pasqua Veronico (p.veronico{at}ba.ipp.cnr.it). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.081679. * Corresponding author; e-mail p.veronico{at}ba.ipp.cnr.it; fax: 39–080–558–0468.
Baracat-Pereira MC, de Almeida Oliveira MG, de Barros EG, Moreira MA, Santoro MM (2001) Biochemical properties of soybean leaf lipoxygenases: presence of soluble and membrane bound forms. Plant Physiol Biochem 39: 91–98[CrossRef] Bleve-Zacheo T, Melillo MT, Zacheo G (1990) Syncytia development in germplasm pea accessions infected with Heterodera goettingiana. Nematol Medit 18: 93–102 Boyington JC, Gaffney BJ, Amzel LM (1993) The three-dimensional structure of an arachidonic acid 15-lipoxygenase. Science 260: 1482–1486 Brash AR (1999) Lipoxygenases: occurrence, function, catalysis, and acquisition of substrate. J Biol Chem 274: 23679–23682 Chang MM, Horovitz D, Culley D, Hadwiger LA (1995) Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding, fungal infection and the elicitor chitosan. Plant Mol Biol 28: 105–111[CrossRef][Web of Science][Medline] Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol 48: 355–381[CrossRef][Web of Science][Medline] Culley DE, Horovitz D, Hadwiger LA (1995) Molecular characterization of disease-resistance response gene DRR206-d from Pisum sativum (L.). Plant Physiol 107: 301–302[CrossRef][Web of Science][Medline] Dellaporta S, Wood J, Hicks J (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21 Di Vito M, Perrino P (1978) Reaction of Pisum spp. to the attacks of Heterodera goettingiana. Nematol Medit 6: 113–118 (in Italian) Domoney C, Firmin JL, Sidebottom C, Ealing PM, Slabas A, Casey R (1990) Lipoxygenase heterogeneity in Pisum sativum. Planta 181: 35–43 Feussner I, Wasternack C (2002) The lipoxygenase pathway. Annu Rev Plant Biol 53: 275–297[CrossRef][Medline] Fukuchi-Mizutani M, Ishiguro K, Nakayama T, Utsunomiya Y, Tanaka Y, Kusumi T, Ueda T (2000) Molecular and functional characterization of a rose lipoxygenase cDNA related to flower senescence. Plant Sci 160: 129–137[Medline] Hornung E, Walther M, Kuhn H, Feussner I (1999) Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis. Proc Natl Acad Sci USA 96: 4192–4197 Hughes RK, Lawson DM, Hornostaj AR, Fairhust SA, Casey R (2001) Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase. Eur J Biochem 268: 1030–1040[Web of Science][Medline] Hughes RK, Wu Z, Robinson DS, Hardy D, West SI, Fairhurst SA, Casey R (1998) Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms. Biochem J 333: 33–43 Ida S, Masaki Y, Morita Y (1983) The isolation of multiple forms and product specificity of rice lipoxygenase. Agric Biol Chem 47: 637–641 Jackson DP (1991) In situ hybridization in plants. In DJ Bowles, SJ Gurr, M McPherson, eds, Molecular Plant Pathology: A Practical Approach. Oxford University Press, Oxford, pp 157–181 Kim E-S, Choi E, Kim Y, Cho K, Lee A, Shim J, Rakwal R, Agrawal GK, Han O (2003) Dual positional specificity and expression of nontraditional lipoxygenase induced by wounding and methyl jasmonate in maize seedlings. Plant Mol Biol 52: 1203–1213[CrossRef][Web of Science][Medline] Leone A, Melillo MT, Bleve-Zacheo T (2001) Lipoxygenase in pea roots subjected to biotic stress. Plant Sci 161: 703–717[CrossRef] Maccarone M, van Zadelhoff G, Veldink GA, Vliegenthart JFG, Finazzi-Agrò A (2000) Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death. Eur J Biochem 267: 5078–5084[Web of Science][Medline] May C, Hohne M, Gnau P, Schwennesen K, Kindl H (2000) The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposome and lipid bodies. Eur J Biochem 267: 1100–1109[Web of Science][Medline] McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4: 1425–1433 Melillo MT, Bleve-Zacheo T, Zacheo G, Perrino P (1990) Morphology and enzyme histochemistry in germplasm pea root attacked by Heterodera goettingiana. Nematol Medit 18: 83–91 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 Å resolution. Biochemistry 35: 10687–10701[CrossRef][Medline] Moy P, Qutob D, Chapman BP, Atkinson I, Gijzen M (2004) Patterns of gene expression upon infection of soybean plants by Phytophthora sojae. Mol Plant Microbe Interact 17: 1051–1062[Web of Science][Medline] Pilet-Nayel L, Muehlbauer FJ, McGee RJ, Kraft JM, Baranger A, Coyne CJ (2002) Quantitative trait loci for partial resistance to Aphanomyces root rot in pea. Theor Appl Genet 106: 28–39[Web of Science][Medline] Porta H, Rocha-Sosa M (2002) Plant lipoxygenases. Physiological and molecular features. Plant Physiol 130: 15–21 Prioul S, Frankewitz A, Deniot G, Morin G, Baranger A (2004) Mapping of quantitative trait loci for partial resistance to Mycosphaerella pinodes in pea (Pisum sativum L.), at the seedling and adult plant stages. Theor Appl Genet 108: 1322–1334[CrossRef][Web of Science][Medline] Ros Barcelo A, Munoz R, Sabater F (1989) Subcellular location of basic and acid soluble isoperoxidases in lupinus. Plant Sci 63: 31–38[CrossRef] Rouet-Mayer MA, Bureau JM, Laurière C (1992) Identification and characterization of lipoxygenase isoforms in senescing carnation petals. Plant Physiol 98: 971–978 Royo J, Leon J, Vancanneyt G, Perez AG, Sanz C, Störmann K, Rosahl S, Sanchez-Serrano JJ (1996) Characterization of three potato lipoxygenases with distinct enzymatic activities and different organ-specific and wound-regulated expression patterns. J Biol Chem 271: 21012–21019 Rusterucci C, Montillet JL, Agnel JP, Battesti C, Alonso B, Knoll A, Bessoule JJ, Etienne P, Suty L, Blein JP, et al (1999) Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves. J Biol Chem 274: 36446–36455 Sambrook J, Fristch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Santino A, De Paolis A, Gallo A, Quarta A, Casey R, Mita G (2003) Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases. Eur J Biochem 270: 4365–4375[Web of Science][Medline] Siedow JN (1991) Plant lipoxygenase: structure and function. Annu Rev Plant Physiol Plant Mol Biol 42: 145–188[CrossRef][Web of Science] Swofford DL (1998) PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods). Sinauer Associates, Sunderland, MA Thompson JP, Greco N, Eastwood R, Sharma SB, Scurrah M (2000) Integrated control of cool food legumes. In R Knight, ed, Linking Research and Marketing Opportunity for Pulses in the 21st Century. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 491–506 Timmerman-Vaughan GM, Frew TJ, Butler R, Murray S, Gilpin M, Falloon K, Johnston P, Lakeman MB, Russel A, Khan T (2004) Validation of quantitative trait loci for Ascochyta blight resistance in pea (Pisum sativum L.), using populations from two crosses. Theor Appl Genet 109: 1620–1631[CrossRef][Web of Science][Medline] Vick BA (1993) Oxygenated fatty acids of the lipoxygenase pathway. In TS Moore Jr, ed, Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, pp 167–191 Wasternack C, Miersch O, Kramell R, Hause B, Ward J, Beale M, Boland W, Parthier B, Feussner I (1998) Jasmonic acid-biosynthesis, signal transduction, gene expression. Fett Lipid 100: 139–146[CrossRef] Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1998) Protein identification and analysis tools in ExPASy server. In AJ Link, ed, Methods in Molecular Biology, 2-D Proteome Analysis Protocols, Vol 112. Humana Press, Totowa, NJ, pp 531–552 Wisniewski JP, Gardner CD, Brewin NJ (1999) Isolation of lipoxygenases cDNA clones from pea nodule mRNA. Plant Mol Biol 39: 775–783[CrossRef][Web of Science][Medline] Zacheo G, Bleve-Zacheo T, Melillo MT (1997) Biochemistry of plant resistance response to nematode infection. In C Fenoll, S Ohl, F Grundler, eds, Cellular and Molecular Aspects of Plant-Nematode Interaction. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 201–213 This article has been cited by other articles:
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ABSTRACT
RESULTS
-factor signal in-frame with LOXN2 coding sequence were selected by PCR control and sequence analysis. For yeast transformation, 10 µg 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. Negative controls included clones containing empty, unmodified vectors. 
