Ferroptosis in Liver Diseases: An Overview.

Ferroptosis is an iron-dependent form of cell death characterized by intracellular lipid peroxide accumulation and redox imbalance. Ferroptosis shows specific biological and morphological features when compared to the other cell death patterns. The loss of lipid peroxide repair activity by glutathione peroxidase 4 (GPX4), the presence of redox-active iron and the oxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids are considered as distinct fingerprints of ferroptosis. Several pathways, including amino acid and iron metabolism, ferritinophagy, cell adhesion, p53, Keap1/Nrf2 and phospholipid biosynthesis, can modify susceptibility to ferroptosis. Through the decades, various diseases, including acute kidney injury; cancer; ischemia-reperfusion injury; and cardiovascular, neurodegenerative and hepatic disorders, have been associated with ferroptosis. In this review, we provide a comprehensive analysis of the main biological and biochemical mechanisms of ferroptosis and an overview of chemicals used as inducers and inhibitors. Then, we report the contribution of ferroptosis to the spectrum of liver diseases, acute or chronic. Finally, we discuss the use of ferroptosis as a therapeutic approach against hepatocellular carcinoma, the most common form of primary liver cancer.

5-Lipoxygenase: Its involvement in gastrointestinal malignancies.

Lipoxygenases (LOXs) are dioxygenases that catalyze the peroxidation of linoleic acid (LA) or arachidonic acid (AA), in the presence of molecular oxygen. The existence of inflammatory component in the tumor microenvironment intimately links the LOXs to gastrointestinal (GI) cancer progression. Amongst the six-different human LOX-isoforms, 5-LOX is the most vital enzyme for leukotriene (LT) biosynthesis, which is the main inflammation intermediaries. As recent investigations have shown the association of 5-LOX with tumor metastasis, there has also been significant progress in discovering the function of 5-LOX pathway in GI cancer. Studies on GI cancer cells using the pharmacological drugs targeting 5-LOX pathway have shown antiproliferative and proapoptotic effects. Pharmacogenetic discoveries in other diseases have revealed strong heritable basis for the leukotriene pathway, which helps in exploring the mechanistic source of genetic alteration within the leukotriene pathway and offer insights into GI cancer pathogenesis and future prospects for treatment and prevention. This review recapitulates the current research status of 5-LOX activity in GI malignancies.

Cancer-produced metabolites of 5-lipoxygenase induce tumor-evoked regulatory B cells via peroxisome proliferator-activated receptor α.

Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report in this study that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase pathway such as leukotriene B4 to activate the peroxisome proliferator-activated receptor α (PPARα) in B cells. Inactivation of leukotriene B4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells and/or tumor 5-lipoxygenase pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

Functional analysis of potato genes involved in quantitative resistance to Phytophthora infestans.

The most significant threat to potato production worldwide is the late blight disease, which is caused by the oomycete pathogen Phytophthora infestans. Based on previous cDNA microarrays and cDNA-amplified fragment length polymorphism analysis, 63 candidate genes that are expected to contribute to developing a durable resistance to late blight were selected for further functional analysis. We performed virus-induced gene silencing (VIGS) to these candidate genes on both Nicotiana benthamiana and potato, subsequently inoculated detached leaves and assessed the resistance level. Ten genes decreased the resistance to P. infestans after VIGS treatment. Among those, a lipoxygenase (LOX; EC 1.13.11.12) and a suberization-associated anionic peroxidase affected the resistance in both N. benthamiana and potato. Our results identify genes that may play a role in quantitative resistance mechanisms to late blight.

Cancer-stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis.

Interactions between cancer cells and stromal cells, including blood vessel endothelial cells (BECs), lymphatic vessel endothelial cells (LECs), bone marrow-derived angiogenic cells (BMDACs) and other bone marrow-derived cells (BMDCs) play important roles in cancer progression. Intratumoral hypoxia, which affects both cancer and stromal cells, is associated with a significantly increased risk of metastasis and mortality in many human cancers. Recent studies have begun to delineate the molecular mechanisms underlying the effect of intratumoral hypoxia on cancer progression. Reduced O2 availability induces the activity of hypoxia-inducible factors (HIFs), which activate the transcription of target genes encoding proteins that play important roles in many critical aspects of cancer biology. Included among these are secreted factors, including angiopoietin 2, angiopoietin-like 4, placental growth factor, platelet-derived growth factor B, stem cell factor (kit ligand), stromal-derived factor 1, and vascular endothelial growth factor. These factors are produced by hypoxic cancer cells and directly mediate functional interactions with BECs, LECs, BMDACs and other BMDCs that promote angiogenesis, lymphangiogenesis, and metastasis. In addition, lysyl oxidase (LOX) and LOX-like proteins, which are secreted by hypoxic breast cancer cells, remodel extracellular matrix in the lungs, which leads to BMDC recruitment and metastatic niche formation.

The investigation into indomethacin-induced potentiation of the contractile response to antigen in ovalbumin-sensitized guinea-pig tracheas.

In the present study, we investigated the mediators involved in the potentiation of antigen-induced contractions by indomethacin in tracheas isolated from ovalbumin (OA)-sensitized guinea-pigs. Indomethacin-induced potentiation of OA contraction was mimicked by prostaglandin DP/EP(1) /EP(2) receptor antagonist, AH-6809 but not by phospholipase A(2) enzyme inhibitor mepacrine. The lipoxygenase inhibitor AA-861 did not affect the contraction response to OA but prevented its potentiation by indomethacin, while the leukotriene receptor antagonist cinalukast inhibited both the OA response and its potentiation. However, the antagonists of platelet-activating factor (PAF) (BN-52021), adenosine (CGS-15943), endothelin ET(A) and ET(B) receptors (BQ-123, BQ-788), and the neutral endopeptidase inhibitor phosphoramidon did not alter the OA-induced contraction and its potentiation by indomethacin. Furthermore, capsaicin and neuropeptide receptor NK1, NK2, and NK3 antagonists (L-732128, MEN-10376, and SB-218795, respectively) also did not affect the OA-induced contractions and its potentiation. On the other hand, the 'transient receptor potential vanilloid 1' (TRPV1) antagonist capsazepine inhibited the potentiation response, while it did not alter the OA contraction itself. In conclusion, the potentiation of OA-induced contraction by indomethacin is more likely due to the increase in lipoxygenase products by the shift of arachidonic acid towards lipoxygenase pathway. Because some of the lipoxygenase products are potent vanilloid agonists, the stimulation of TRPV1 receptors besides leukotriene receptors seems to participate in the potentiation of contraction response in sensitized guinea-pig tracheas. PAF, adenosine, endothelins, and the neuropeptides present in the afferent neurons do not contribute to the potentiation of OA-induced contraction by indomethacin.

PDGF-BB and TGF-{beta}1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress.

Shear stress, especially low shear stress (LowSS), plays an important role in vascular remodeling during atherosclerosis. Endothelial cells (ECs), which are directly exposed to shear stress, convert mechanical stimuli into intracellular signals and interact with the underlying vascular smooth muscle cells (VSMCs). The interactions between ECs and VSMCs modulate the LowSS-induced vascular remodeling. With the use of proteomic analysis, the protein profiles of rat aorta cultured under LowSS (5 dyn/cm(2)) and normal shear stress (15 dyn/cm(2)) were compared. By using Ingenuity Pathway Analysis to identify protein-protein association, a network was disclosed that involves two secretary molecules, PDGF-BB and TGF-ß1, and three other linked proteins, lamin A, lysyl oxidase, and ERK 1/2. The roles of this network in cellular communication, migration, and proliferation were further studied in vitro by a cocultured parallel-plate flow chamber system. LowSS up-regulated migration and proliferation of ECs and VSMCs, increased productions of PDGF-BB and TGF-ß1, enhanced expressions of lysyl oxidase and phospho-ERK1/2, and decreased Lamin A in ECs and VSMCs. These changes induced by LowSS were confirmed by using PDGF-BB recombinant protein, siRNA, and neutralizing antibody. TGF-ß1 had similar influences on ECs as PDGF-BB, but not on VSMCs. Our results suggest that ECs convert the LowSS stimuli into up-regulations of PDGF-BB and TGF-ß1, but these two factors play different roles in LowSS-induced vascular remodeling. PDGF-BB is involved in the paracrine control of VSMCs by ECs, whereas TGF-ß1 participates in the feedback control from VSMCs to ECs.

[Roles of lipid mediators in controlling vascular inflammation and the progression of atherosclerosis].

Atherosclerosis is recognized as an inflammatory condition of the vessel wall, characterized by accumulation of inflammatory cells such as macrophages and T cells. There are accumulating evidences that chemokines, cytokines, and lipid mediators coordinately modulate platelet- or leukocyte-endothelial cell interactions, and contribute to the maintenance of vascular homeostasis. This review focuses on the role of lipid mediators, especially those derived from polyunsaturated fatty acids, in controlling vascular inflammation and the progression of atherosclerosis.

AmbLOXe--an epidermal lipoxygenase of the Mexican axolotl in the context of amphibian regeneration and its impact on human wound closure in vitro.

OBJECTIVE: The Mexican axolotl (Ambystoma mexicanum) is a well-characterized example for intrinsic regeneration. As lipoxygenase signaling is of crucial importance to scarless mammalian wound healing, we postulated that lipoxygenases might be expressed during amphibian regeneration and they might also influence human cells under appropriate conditions. In this study we identified an amphibian lipoxygenase and evaluated its impact on human cells in an in vitro wound model. METHODS: cDNA encoding for amphibian epidermal lipoxygenase (AmbLOXe) was polymerase chain reaction amplified and sequenced followed by phylogenic classification based on T-coffee alignment. Distribution of AmbLOXe was examined in various Ambystoma tissues, using polymerase chain reaction and in situ hybridization. Lipoxgenase influence was investigated using an outgrowth model of amphibian epidermal cells. Human osteosarcoma, as well as keratinocyte cell lines expressing AmbLOXe, were tested concerning in vitro wound closure in a monolayer scratch model. RESULTS: We isolated AmbLOXe from Ambystoma limb bud blastema identified as a homologue of human epidermal lipoxygenase. Amphibian epidermal lipoxygenase is expressed in Axolotl limb blastema and in epidermal cells which show decreased cell migration and proliferation rates when treated with LOX inhibitors. Furthermore, human osteosarcoma and keratinocyte cells showed increased rates of cell migration if transfected with AmbLOXe. CONCLUSION: In this study, AmbLOXe, a new effector of amphibian regeneration is described. In consideration of the presented data, AmbLOXe is important for amphibian epidermal cell proliferation and migration. As AmbLOXe expressing human osteosarcoma and keratinocyte cell lines showed increased rates of in vitro wound closure, an influence of amphibian mediators on human cells could be described for the first time.

Mono(2-ethylhexyl) phthalate induces both pro- and anti-inflammatory responses in rat alveolar macrophages through crosstalk between p38, the lipoxygenase pathway and PPARalpha.

Airway inflammation is important in asthma pathogenesis. Recent epidemiological data have indicated an association between asthma symptoms in children and exposure to di(2-ethylhexyl) phthalate (DEHP). Thus, we have studied inflammatory responses in primary rat alveolar macrophages (AMs) after exposure to mono(2-ethylhexyl) phthalate (MEHP), the major primary metabolite of DEHP. First, we show that MEHP induces a dose-dependent release of the pro-inflammatory tumour necrosis factor-alpha (TNF-alpha) in AMs, giving a maximal (5-fold) increase at 0.7 mM. This concentration also induced some cell death. MEHP also induced phosphorylation of MAPK p38, while the p38 inhibitor SB 202190 reduced MEHP-induced TNF-alpha, suggesting a p38-dependent cytokine production. Next, we elucidated possible effects of MEHP on the 5-lipoxygenase (5-LO) pathway and found that MEHP caused increased leukotriene (LTB(4)) release. Further, we found that the 5-LO inhibitor nordihydrogualaretic acid (NDGA) significantly reduced both MEHP-induced TNF-alpha release and MEHP-induced formation of reactive oxygen species (ROS), supporting an involvement of the 5-LO pathway in MEHP induced inflammatory reactions. Last, we found that MK-886, a known inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), increased the MEHP-induced TNF-alpha response. This indicates that MEPH-PPARalpha binding mediates an anti-inflammatory signal.

Cyclooxygenases and the pathogenesis of chronic rhinosinusitis and nasal polyposis.

Cyclooxygenase (COX) enzymes catalyze the rate-limiting steps in prostaglandin synthesis. Prostaglandins have an important role in several physiological processes such as maintenance of gastrointestinal integrity and pathological processes such as inflammation and neoplasia. Several mechanisms have been proposed for the development of chronic rhinosinusitis, but the common final pathway seems to be an integrated process involving the mucosal epithelium, matrix, and inflammatory cells and mediators. Upper and lower airway pathologies coexist and share common etiopathogenic mechanisms, and nasal polyposis is often associated with asthma and aspirin sensitivity. The cellular source of COX activity in acute and chronic inflammation, as in chronic rhinosinusitis, is poorly understood. COX theory postulated that inhibition of COX broke down biochemical reactions that lead to the development of asthma attacks. This article focuses on COX in the pathogenesis of chronic rhinosinusitis and nasal polyposis.

Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin.

Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells.

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

Effect of the interaction between lipoxygenase pathway and progesterone on the regulation of hydroxysteroid 11-Beta dehydrogenase 2 in cultured human term placental trophoblasts.

Placental hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2) plays an important role in pregnancy maintenance and fetal maturation. In the event of intrauterine infection, lipoxygenase (LOX) metabolites are produced in the placenta and contribute to preterm labor and adverse fetal outcomes. On the other hand, LOX metabolites are involved in production of progesterone, which is required for pregnancy maintenance. In this study, we evaluated the interaction between the LOX pathway, progesterone, and HSD11B2. Specifically, we hypothesized that LOX metabolites would alter HSD11B2 and this effect would be mediated by progesterone. We cultured human term placental trophoblasts in the presence and absence of the LOX inhibitors Nordihydroguaiaretic acid (NDGA), AA861, and Baicalein; the LOX metabolites Leukotriene B(4) and 12(S)-Hydroxyeicosatetraenoate (12-HETE); and progesterone and progesterone receptor antagonist RU486. By radiometric conversion assay, real-time quantitative PCR, Western blot analysis, and ELISA, we examined HSD11B2 enzyme activity, HSD11B2 mRNA and HSD11B2 protein expression, and progesterone output. LOX metabolites down-regulated HSD11B2 activity and HSD11B2 expression. LOX inhibitors up-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were attenuated by addition of LOX metabolites. Net progesterone output was increased by LOX metabolites and decreased by LOX inhibitors. Progesterone down-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were blocked by RU486. Furthermore, the suppressive effect of 12-HETE on HSD11B2 activity was also reversed by RU486. We conclude that HSD11B2 in human placental trophoblasts is decreased by progesterone and increased by inhibition of endogenous LOX metabolites, and that a component of the effect of LOX metabolites on HSD11B2 is mediated by their stimulation of endogenous progesterone output.

Lipoxygenase metabolism: roles in tumor progression and survival.

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies.

Regulation of COX and LOX by curcumin.

Turmeric (Curcuma longa) is extensively used as a household remedy for various diseases. For the last few decades, work has been done to establish the biological activities and pharmacological actions of curcumin, the principle constituent of turmeric. Curcumin has proven to be beneficial in the prevention and treatment of a number of inflammatory diseases due to its anti-inflammatory activity. Arachidonic acid-derived lipid mediators that are intimately involved in inflammation are biosynthesized by pathways dependent on cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. The role of LOX and COX isoforms, particularly COX-2, in the inflammation has been well established. At cellular and molecular levels, curcumin has been shown to regulate a number of signaling pathways, including the eicosanoid pathway involving COX and LOX. A number of studies have been conducted that support curcumin-mediated regulation of COX and LOX pathways, which is an important mechanism by which curcumin prevents a number of disease processes, including the cancer. The specific regulation of 5-LOX and COX-2 by curcumin is not fully established; however, existing evidence indicates that curcumin regulates LOX and COX-2 predominately at the transcriptional level and, to a certain extent, the posttranslational level. Thus, the curcumin-selective transcriptional regulatory action of COX-2, and dual COX/LOX inhibitory potential of this naturally occurring agent provides distinctive advantages over synthetic COX/LOX inhibitors, such as nonsteroidal anti-inflammatory drugs. In this review, we discuss evidence that supports the regulation of COX and LOX enzymes by curcumin as the key mechanism for its beneficial effects in preventing various inflammatory diseases.

Cyclooxygenases and lipoxygenases in prostate and breast cancers.

Oxidative metabolism of polyunsaturated fatty acids through cyclooxygenases or lipoxygenases can generate various lipid peroxides and bioactive lipids, and regulate cellular proliferation, apoptosis, differentiation and senescence. The role of the second cyclooxygenase isoform (COX-2) has been demonstrated in a number of studies and is regarded as a promising target for chemoprevention and treatment. The involvement for lipoxygenases in tumor initiation and progression has been implicated in several studies but remains controversial. Among the many members of lipoxygenase family, both tumor promoting and suppressing activities have been described. For example, 15-lipoxygenase-1 has been implicated as a tumor promoter in prostate cancer, but it suppresses colon cancer. In this review, the role of cyclooxygenases and lipoxygenases in cancer will be described, with the hope of attracting further research to define their functions in cancer.

Characterization of a divinyl ether biosynthetic pathway specifically associated with pathogenesis in tobacco.

In tobacco (Nicotiana tabacum), an elicitor- and pathogen-induced 9-lipoxygenase (LOX) gene, NtLOX1, is essential for full resistance to pathogens, notably to an incompatible race of Phytophthora parasitica var. nicotianae (Ppn race 0). In this work, we aimed to identify those oxylipins induced during attempted infection by Ppn race 0 and down-regulated in NtLOX1 antisense plants. Here we show that colneleic and colnelenic acids, which significantly inhibit germination of Ppn zoospores, are produced in roots of wild-type plants inoculated with Ppn, but are down-regulated in NtLOX1 antisense plants. A search for a tobacco gene encoding the enzyme involved in the formation of these divinyl ether (DVE) fatty acids resulted in the cloning and characterization of a DVE synthase (DES) clone (NtDES1). NtDES1 is a 9-DES, specifically converting fatty acid 9-hydroperoxides into DVE fatty acids. NtDES1 has the potential to act in combination with NtLOX1 because, in the presence of the two enzymes, linoleic and linolenic acids were converted in vitro into colneleic and colnelenic acids, respectively. In addition, the pattern of NtDES1 gene expression was quite similar to that of NtLOX1. Their transcripts were undetected in healthy tissues from different plant organs, and accumulated locally and transiently after elicitation and fungal infection, but not after wounding. Visualization of NtDES1-yellow fluorescent protein and NtLOX1-cyan fluorescent protein fusion proteins in tobacco leaves indicated that both localize in the cytosol and are excluded from plastids, consistent with the presumed location of the 9-LOX pathway in plants and the lack of transit peptides for NtLOX1 and NtDES1, respectively. Our data suggest that, in tobacco, NtDES1 and NtLOX1 act together and form DVEs in response to pathogen attack and that this class of oxylipins modulates in vivo the outcome of the tobacco-Ppn race 0 interaction.

A novel lipoxygenase in pea roots. Its function in wounding and biotic stress.

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.