Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures. Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin. Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation. The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction. These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation. Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation. These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation. Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+). Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production.     

The Lipoxygenase Pathway is Involved in Elicitor-induced Phytoalexin Accumulation in Plane Tree (Platanus acerifolia) Cell-suspension Cultures

The Ceratocystis fimbriata f.sp. platani 66 kDa glycoprotein elicitor-induced secretion of soluble coumarins by plane tree (Platanus acerifolia (Aiton) Wild) cell-suspension cultures was investigated by studying the possible involvement of the octadecanoid pathway in the cell response. When cell-suspension cultures were treated with the glycoprotein elicitor, the cells exhibited a rapid and transient increase in lipoxygenase activity, in synthesis of endogenous jasmonic acid prior to the accumulation of coumarin phytoalexins. The treatment of cells with an inhibitor of lipoxygenase (ETYA) before elicitor addition, drastically reduced the lipoxygenase activity, the production of endogenous jasmonic acid and phytoalexin accumulation. The results demonstrate the role of the jasmonate pathway in the intracellular signal cascade.     

Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.     

Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.     

脂氧合酶在诱导红豆杉细胞产紫杉醇中的作用

对红豆杉悬浮培养细胞中脂氧合酶(LOX)在诱导子诱导紫杉醇合成中的作用进行了探讨。结果表明真菌诱导子处理可提高细胞内LOX的活性和紫杉醇的产量,而诱导前用LOX抑制剂菲尼酮处理,可完全抑制诱导子对LOX活性和紫杉醇合成的诱导作用。说明LOX途径可能参与了紫杉醇的合成过程。外加茉莉酸甲酯也可激活LOX活性和紫杉醇合成,诱导前用菲尼酮处理可抑制诱导子诱导的LOX活性和紫杉醇合成,说明外源茉莉酸甲酯可能是通过激活细胞内LOX途径而启动下游紫杉醇的合成。为了进一步研究脂氧合酶在紫杉醇合成中的作用。我们还对红豆杉细胞脂氧合酶的分布和分子量等性质进行了研究。     

Synergistic stimulation of S-adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1-oleoyl-2-acetylglycerol

The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h. These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Evaluation of the yeast-extract signaling pathway leading to silymarin biosynthesis in milk thistle hairy root culture

The biosynthesis of silymarin, a potent antihepatotoxic compound, from the dried fruits of Silybum marianum L. Gaertn in hairy root cultures can be stimulated by a yeast extract elicitor. These results correlated with culture time, and the biosynthesis reached a maximum of 0.47 mg g^?1 DW by 72 h after culture (2-fold higher than the control). Lipoxygenase activity and linoleic acid content were stimulated by this treatment, suggesting that the jasmonate pathway may mediate the elicitor-induced accumulation of silymarin. The H₂O₂ content increased 24 h after elicitation and did not have marked changes between 48 and 72 h. In addition, the tocopherol content (especially α- and δ-tocopherols) increased 72 h after elicitation in comparison with non-treated cultures. Ascorbate had trace changes during feeding time and was lower than the control. The antioxidant activity was assayed by the 1-1-diphenyl-2-picrylhydrazyl stable free radical method and results were calculated base on an IC₅₀ that increased upon treatment, especially 24 h after treatment, with changes related to H₂O₂ content. These observations suggested that reactive oxygen species may mediate elicitor signals to the jasmonate pathway that lead to the production of silymarin.     

Lysophosphatidylserine stimulates leukemic cells but not normal leukocytes

In this study, we observed that lysophosphatidylserine (LPS) stimulated intracellular calcium ([Ca(2+)](i)) increase in leukemic cells but not in normal human peripheral blood mononuclear cells. LPS also stimulated [Ca(2+)](i) increase in human leukemic THP-1 cells. LPS-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343. LPS also stimulated inositol phosphates formation in THP-1 cells, suggesting that LPS stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) completely inhibited [Ca(2+)](i) increase by LPS, indicating the activation of PTX-sensitive G-proteins. We also found that LPS-induced [Ca(2+)](i) increase was completely inhibited by suramin, suggesting G-protein coupled receptor activation. Since LPS specifically stimulates PTX-sensitive G-proteins, phospholipase C-dependent [Ca(2+)](i) increase in leukemic cells but not normal peripheral blood leukocytes, LPS receptor may be associated with leukemia.     

Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.     

Pharmacological characterisation of neuropeptide F (NPF)-induced effects on the motility of Mesocestoides corti (syn. Mesocestoides vogae) larvae

Neuropeptide F is the most abundant neuropeptide in parasitic flatworms and is analogous to vertebrate neuropeptide Y. This paper examines the effects of neuropeptide F on tetrathyridia of the cestode Mesocestoides vogae and provides preliminary data on the signalling mechanisms employed. Neuropeptide F (>/=10 microM) had profound excitatory effects on larval motility in vitro. The effects were insensitive to high concentrations (1 mM) of the anaesthetic procaine hydrochloride suggesting extraneuronal sites of action. Neuropeptide F activity was not significantly blocked by a FMRFamide-related peptide analog (GNFFRdFamide) that was found to inhibit GNFFRFamide-induced excitation indicating the occurrence of distinct neuropeptide F and FMRFamide-related peptide receptors. Larval treatment with guanosine 5'-O-(2-thiodiphosphate) trilithium salt prior to the addition of neuropeptide F completely abolished the excitatory effects indicating the involvement of G-proteins and a G-protein coupled receptor in neuropeptide F activity. Addition of guanosine 5'-O-(2-thiodiphosphate) following neuropeptide F had limited inhibitory effects consistent with the activation of a signalling cascade by the neuropeptide. With respect to Ca(2+) involvement in neuropeptide F-induced excitation of M. vogae larvae, the L-type Ca(2+)-channel blockers verapamil and nifedipine both abolished neuropeptide F activity as did high Mg(+) concentrations and drugs which blocked sarcoplasmic reticulum Ca(2+)-activated Ca(2+)-channels (ryanodine) and sarcoplasmic reticulum Ca(2+) pumps (cyclopiazonic acid). Therefore, both extracellular and intracellular Ca(2+) is important for neuropeptide F excitation in M. vogae. With respect to second messengers, the protein kinase C inhibitor chelerythrine chloride and the adenylate cyclase inhibitor MDL-2330A both abolished neuropeptide F-induced excitation. The involvement of a signalling pathway that involves protein kinase C was further supported by the fact that phorbol-12-myristate-13-acetate, known to directly activate protein kinase C, had direct excitatory effects on larval motility. Although neuropeptide F is structurally analogous to neuropeptide Y, its mode-of-action in flatworms appears quite distinct from the common signalling mechanism seen in vertebrates.     

Distinct pathways for jasmonate- and elicitor-induced expressions of a cytochrome P450 gene in soybean suspension-cultured cells

The mechanisms of the jasmonate-induced expression of genes encoding the cytochrome P450 CYP93A1 and lipoxygenase L-4 were analyzed in a soybean photomixotrophic cultured cell line, SB-P. The induction of the cytochrome P450 gene caused by methyl jasmonate (MeJA) was specifically suppressed by trifluoperazine and DCMU, inhibitors of chloroplast electron transport. Additionally, induction of the cytochrome P450 gene required irradiation. In contrast, induction of the lipoxygenase L-4 gene by the MeJA treatment occurred in both dark and light. Based on the results, the presence of two distinct signalling pathways for jasmonate-inducible gene expression, light-dependent and light-independent, is proposed. The jasmonate-inducible cytochrome P450 was also specifically induced by a fungal elicitor from a cell wall fraction of Phytophthora megasperma , a fungal pathogen, suggesting a role for P450 in the defense response to fungus in soybean cells. However, trifluoperazine did not block the elicitor-induced expression of cytochrome P450.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Induction and signaling of an apoptotic response typified by DNA laddering in the defense response of oats to infection and elicitors

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.     

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.