Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont.) deBary

The dose response for elicitation of the hypersensitive reaction in potato tuber discs by arachidonic acid (AA) suggested saturation at higher concentrations. Glucans from Phytophthora infestans, inactive themselves as elicitors of the hypersensitive reaction, enhanced sesquiterpene accumulation and hypersensitive browning elicited by AA. Significant activity (seven times control values) was observed with 33 pmol AA/3.0-cm potato disc in the presence of glucans. Glucans did not affect accumulation of steroid glycoalkaloids, influence the timing or relative amounts of sesquiterpenes which accumulate, or affect recovery of AA added to potato discs. Glucans enhanced activity whether added to potato discs 18 h prior to AA, at the same time as AA, or 18 h after AA. Elicitor activity in the presence of glucans was evident with 20-carbon unsaturated fatty acids that had little or no elicitor activity in the absence of glucans. The position of double bonds had considerable influence on the specific activity of unsaturated fatty acids. The most active had a minimum of three double bonds in a methylene-interrupted series beginning with delta 5, e.g., delta 5,8,11. A delta 5 double bond conferred significant activity even if it was not part of a methylene-interrupted series. The 20-carbon chain length appeared optimal for elicitor activity. The 22-carbon chain acids had low activity, and 16- and 18-carbon acids were inactive. A free carboxyl group or easily transesterified group appeared necessary for activity. Arachidonyl alcohol had very low activity and arachidonyl cyanide was inactive. AA-containing phosphatidylcholine, lysophosphatidylcholine and monoacylglycerol were at least as active as free AA, AA-containing diacylglycerols were slightly less active than free AA, and triarachidonyl glycerol was inactive.     

Regulation of tobacco lipoxygenase by methyl jasmonate and fatty acids

Lipoxygenase (LOX) activity and gene expression have been described previously to be induced in tobacco by fungal infection and elicitor treatment. We now report that LOX activity is induced in tobacco cell suspensions by treatment with methyl jasmonate (MeJa). This compound had no effect on the in vitro activity of tobacco LOX. Induction of LOX activity is a dose-dependent response with a maximum around 890 μM MeJa. Linolenic acid, the precursor for jasmonate synthesis, also induces LOX activity. When applied together with fungal elicitor, linolenic acid drastically increases and prolongs the induction of LOX activity. LOX activity and gene expression in elicited tobacco cells are partially inhibited by pretreatment with eicosatetraynoic acid (ETYA), a potent inhibitor of tobacco LOX in vitro. The induction by methyl jasmonate, in contrast, was not inhibited by ETYA pretreatment. These data suggest that induction of LOX gene expression and activity upon elicitation are regulated at least partially by LOX products. © Académie des Sciences/ Elsevier, Paris     

A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads

Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR.     

Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor,Chitin Oligosaccharides,Chitosan, and Methyl Jasmonate

A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.C. 1.13.11.12) in wheat (Triticum aestivum) leaves. Immunoblot analysis using anti-LOX antibodies revealed the induction of 92- and 103-kD LOX species after Pgt elicitor treatment. In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants. The effects of chitin oligomers and chitosan resembled those caused by MJ. In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action. LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions. It is most active at pH 5.5 to 6.0, and product formation from linoleic and [alpha]-linolenic acid is clearly in favor of the 9-LOOHs. It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves.     

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.     

Pseudoperoxidase investigations of hydroperoxides and inhibitors with human lipoxygenases

Understanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234 nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result.     

Characterization of a membrane-associated apoplastic lipoxygenase in Phaseolus vulgaris L.

An extracytoplasmic 86.7 kDa protein was isolated from intercellular washing fluids (IWF) of Phaseolus vulgaris etiolated hypocotyls. Micro sequencing of tryptic peptides of the 86.7 kDa protein revealed 100% identity with a bean lipoxygenase (LOX) protein fragment. Purified P87-LOX exhibited LOX activity characterized by an optimal pH of 6.0 and linolenic acid as an optimal substrate, and was classified as a 13-LOX with respect to its positional specificity of linoleic acid oxygenation. A protein identical to P87-LOX, as determined by MALDI-TOF analysis and biochemical characterization, was purified from hypocotyl microsomes. Immunoblot analysis showed that P87-LOX is present in plasma membrane-enriched fractions, from which it was solubilized using high ionic strength buffers. These observations suggest that P87-LOX is a peripheral protein associated to the apoplastic face of the plasma membrane.     

Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

COX-2/12-LOX dual pathway, a novel strategy for treatment of multiple myeloma?

Cyclooxygenase (COX) and lipoxygenase (LOX) metabolic enzymes are the two main pathways for arachidonic acid (AA) metabolism. Emerging reports now indicate alterations of arachidonic acid metabolism with carcinogenesis and many COX and LOX inhibitors are being investigated as potential anticancer drugs. COX-2 is frequently expressed in many tumors, such as multiple myeloma (MM), a disorder in which malignant plasma cells accumulate, generally derived from one clone in the bone marrow, and is an independent predictor of poor outcome. 12-LOX, an important member of LOX, is proved to be expressed in MM cells. We hypothesize that COX-2 and 12-LOX represent an integrated system, COX-2/12-LOX dual pathway, which much more efficiently enhances the intracellular levels of unesterified arachidonate and regulates cell proliferative, apoptosis and pro-angiogenic potential of MM. The COX-2/12-LOX dual pathway may act as a novel potential strategy for treatment of tumors co-expressing COX-2 and 12-LOX, and the agents that can simultaneously inhibit the two enzymes of COX-2 and 12-LOX may present a novel and promising therapeutic approach to these tumors.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

Early Responses in Methyl Jasmonate-Preconditioned Cells toward Pathogen-Derived Elicitors

Early, signal transduction-related responses in cultured tobacco cells due to methyl jasmonate (MeJa), a cell-wall-derived elicitor from Phytophthora nicotianae and chitosan, were investigated. MeJa was an effective inducer of lipid peroxidation and lipoxygenase (LOX) activity with maximum levels reached within 2 h and 4–8 h, respectively. Chitosan and the elicitor induced a transient increase (1–4 h) in lipid peroxidation. Conditioning with MeJA, followed by secondary elicitation, led to a significant increase in malondialdehyde concentration after 1 h. Chitosan and the elicitor induced transient activation of LOX with maximal values between 8 and 12 h, with preconditioning resulting in a rapid increase in LOX activity at 4 h post elicitation. MeJA did not effect phosphoprotein accumulation but conditioning led to the potentiation and differential induction of phosphoproteins due to chitosan and elicitor. The results indicate that cells are sensitized by the exposure to MeJa to respond more intensely and rapidly toward secondary elicitation by fungal pathogen derived elicitors.     

Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum

Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g^–1 dry wt) was the most predominant followed by lubimin (171 g g^–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites.     

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 μg g^?1 dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 μg g^?1 dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.     

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.     

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions. Received: 4 July 1996 / Accepted: 23 November 1996     

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL,Cht-1, and PBZ1, but not of Lox in rice

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.     

Fatty acid oxidation and signaling in apoptosis

It is well established that fatty acid metabolites of cyclooxygenase, lipoxygenase (LOX), and cytochrome P450 are implicated in essential aspects of cellular signaling including the induction of programmed cell death. Here we review the roles of enzymatic and non-enzymatic products of polyunsaturated fatty acids in controlling cell growth and apoptosis. Also, the spontaneous oxidation of polyunsaturated fatty acids yields reactive aldehydes and other products of lipid peroxidation that are potentially toxic to cells and may also signal apoptosis. Significant conflicting data in terms of the role of LOX enzymes are highlighted, prompting a re-evaluation of the relationship between LOX and prostate cancer cell survival. We include new data showing that LNCaP, PC3, and Du145 cells express much lower levels of 5-LOX mRNA and protein compared with normal prostate epithelial cells (NHP2) and primary prostate carcinoma cells (TP1). Although the 5-LOX activating protein inhibitor MK886 killed these cells, another 5-LOX inhibitor AA861 hardly showed any effect. These observations suggest that 5-LOX is unlikely to be a prostate cancer cell survival factor, implying that the mechanisms by which LOX inhibitors induce apoptosis are more complex than expected. This review also suggests several mechanisms involving peroxisome proliferator activated receptor activation, BCL proteins, thiol regulation, and mitochondrial and kinase signaling by which cell death may be produced in response to changes in non-esterified and non-protein bound fatty acid levels. Overall, this review provides a context within which the effects of fatty acids and fatty acid oxidation products on signal transduction pathways, particularly those involved in apoptosis, can be considered in terms of their overall importance relative to the much better studied protein or peptide signaling factors.     

Pharmacodynamics simulation of HOEC by a computational model of arachidonic acid metabolic network

Background: Arachidonic acid (AA) metabolic network is activated in the most inflammatory related diseases, and small-molecular drugs targeting AA network are increasingly available. However, side effects of above mentioned drugs have always been the biggest obstacle. (+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC), a natural product acted as an inhibitor of 5-lipoxygenase (5-LOX) and 15-LOX in vitro, exhibited weaker therapeutic effect in high dose than that in low dose to collagen induced arthritis (CIA) rats. In this study, we tried to elucidate the potential regulatory mechanism by using quantitative pharmacology. Methods: First, we generated an experimental data set by monitoring the dynamics of AA metabolites’ concentration in A23187 stimulated and different doses of HOEC co-incubated RAW264.7. Then we constructed a dynamic model of A23187-stimulated AA metabolic model to evaluate how a model-based simulation of AA metabolic data assists to find the most suitable treatment dose by predicting the pharmacodynamics of HOEC. Results: Compared to the experimental data, the model could simulate the inhibitory effect of HOEC on 5-LOX and 15-LOX, and reproduced the increase of the metabolic flux in the cyclooxygenase (COX) pathway. However, a concomitant, early-stage of stimulation-related decrease of prostaglandins (PGs) production in HOEC incubated RAW264.7 cells was not simulated in the model. Conclusion: Using the model, we predict that higher dose of HOEC disrupts the flux balance in COX and LOX of the AA network, and increased COX flux can interfere the curative effects of LOX inhibitor on resolution of inflammation which is crucial for the efficient and safe drug design.