Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.     

Cerium elicitor-induced phosphatidic acid triggers apoptotic signaling development in Taxus cuspidata cell suspension cultures

Degradation of membrane phospholipids is associated with apoptotic responses, but the signaling development of this degradation is not well understood. Cerium (Ce⁴⁺), an important rare earth element, induces cellular apoptosis and taxol biosynthesis in Taxus cuspidata suspension cultures. Here, using mass spectrometry and biochemical technique, we demonstrated that the phospholipase D (PLD) was rapidly activated by Ce⁴⁺ and hydrolyzed structural phospholipids to generate lipid signal molecule, phosphatidic acid (PA). 1-Butanol, an antagonist of PLD-dependent PA production, blocked the biphasic burst of superoxide anions (O₂⁻) and thus mitigated cellular apoptosis. The time-course analysis of PA accumulation and ERK-like mitogen-activated protein kinase (MAPK) regulation indicated PA generation preceded MAPK activation, suggesting that the rapid accumulation of PA might be required for the initial MAPK activity. After 2 h of Ce⁴⁺ elicitation, however, PA-induced O₂⁻ burst, forming a negative regulation to MAPK activity, which in turn led to apoptotic signaling development.     

Comparative characteristics of the metabolism of E. coli cells with various activity of penicillin acylase

A correlation between the synthesis and secretion of penicillin acylase (PA; EC 3.5.1.11) and the membrane phospholipid composition was observed in three E. coli strains. In cells with overproduction of PA, the phospholipid/protein ratio decreases, while the cardiolipin/phosphatidylglycerol ratio increases. The differences in the functioning of the electron transport system were revealed in cells with different levels of PA synthesis and secretion. The O2 consumption rate was 3 times lower in the cells with overproduction of PA than in those of less productive strains. On the contrary, membrane particles isolated from the cells of PA producers had no significant differences in the O2-reduction rate. The sensitivity of the strains to the inhibitor of terminal oxidases, sodium cyanide, and to the uncoupler of redox phosphorylation, chlorocarbonyl-phenylhydrazone, was different. Thus the E. coli cells with PA overproduction are characterized by significant changes in energetics and constructive metabolism. The interrelations between PA overproduction, phospholipid metabolism and the respiratory chain activity are discussed.     

Activation of Phospholipase A by Plant Defense Elicitors

Participation of phospholipase A (PLase A) in plant signal transduction has been documented for auxin stimulation of growth but not for elicitation of any plant defense response. In this paper, we report two independent assays for monitoring PLase A induction in plant cells and have used these assays to evaluate whether transduction of defense-related signals might require PLase A activation. Oligogalacturonic acid, a potent elicitor of the soybean (Glycine max) H2O2 burst, was unable to stimulate endogenous PLase A, suggesting that PLase A activation is not an obligate intermediate in the oligogalacturonic acid-induced burst pathway. In contrast, harpin and an extract from the pathogenic fungus Verticillium dahliae both stimulated the oxidative burst and promoted a rapid increase in PLase A activity. To evaluate the possible role of this inducible PLase A activity in transducing the oxidative burst, we tested the effect of chlorpromazine-HCl, a PLase A inhibitor on elicitor-stimulated burst activity. Pretreatment with chloropromazine was found to inhibit the H2O2 burst triggered by V. dahliae extract at the same concentration at which it blocked PLase A activation. In contrast, neither the harpin- nor oligogalacturonic acid-induced burst was altered by addition of chlorpromazine. These data suggest that PLase A stimulation may be important in certain elicitor-induced oxidative bursts (e.g. V. dahliae) and that other elicitors such as oligogalacturonic acid and harpin must operate through independent signaling intermediates to activate the same defense response.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

Lipidomic analysis reveals differential defense responses of Taxus cuspidata cells to two elicitors, methyl jasmonate and cerium (Ce4+)

Methyl jasmonate (MeJA) and cerium (Ce(4+)) elicitation share common features of increasing taxol accumulation of Taxus cuspidata cells. Interestingly, Ce(4+) induces programmed cell death (PCD), but this phenomenon is not observed with MeJA elicitation. Here, using a lipidomic approach to measure more than 100 membrane glycerophospholipids of T. cuspidata cells quantitatively, we discovered that lysophosphatidylcholine (LysoPC), phosphatidic acid (PA) and phosphatidylcholine were three potential lipid markers that were responsible for the differences between Ce(4+)-induced cells and MeJA-induced cells. Compared with MeJA elicitation, marked increase of phospholipase D (PLD) activity was observed following Ce(4+) elicitation, suggesting that the PLD activation and high concentrations of PA production might mediate the PCD. Rapid increase of phospholipase A(2) (PLA(2)) activity caused the release of fatty acids and LysoPC following Ce(4+) elicitation, which enhanced endogenous jasmonic acid (JA) accumulation. In contrast, PLA(2) activity was poorly induced following MeJA elicitation. PLA(2) inhibitor suppressed not only JA accumulation but also taxol production, suggesting that the PLA(2) activation mediated Ce(4+)-induced taxol production partially through a JA-dependent signaling pathway. These results demonstrate that differential alternation of glycerolphospholipids caused by phospholipases constitutes an important step in cell death response to Ce(4+) and increasing taxol production.     

Exopolysaccharides of Pantoea agglomerans have different priming and eliciting activities in suspension-cultured cells of monocots and dicots

Induced disease resistance of plants is often associated with an enhanced capacity to activate cellular defense responses to pathogen attack, named the "primed" state of the plant. Exopolysaccharides of Pantoea agglomerans have recently been reported as the first priming active component of bacterial origin in wheat cells. We now show that Pantoea exopolysaccharides also prime rice cells for better elicitation of a rapid oxidative burst. In contrast, in tobacco and parsley cell cultures Pantoea exopolysaccharides activate the oxidative burst response directly. Our results point to a different recognition and/or mode of action of Pantoea exopolysaccharides in monocot and dicot plants.     

Diacylglycerol kinases activate tobacco NADPH oxidase‐dependent oxidative burst in response to cryptogein

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial‐associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [³³P]‐orthophosphate labelling of tobacco Bright Yellow‐2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide‐dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD‐mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH‐oxidase activity. Amongst cluster III DGKs, the expression of DGK5‐like was up‐regulated in response to cryptogein. Besides DGK5‐like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid‐mediated events in plant immunity.     

Unsaturated fatty acids as second messengers of superoxide generation by macrophages

Chemically elicited guinea pig peritoneal exudate macrophages respond by superoxide (O2-) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O2- production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca2+. O2- production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O2- production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O2- production by stimulants or fatty acids. On the contrary, O2- formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O2- generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O2- production; native and lipoxygenase-treated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O2- production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O2- generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.     

Cross-talk of polyamines and nitric oxide in endophytic fungus-induced betulin production in Betula platyphylla plantlets

Key message

This paper showed that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.

Abstract

The aim of this study was to investigate the relationship between nitric oxide (NO) and polyamines (PAs) and to determine their roles in betulin accumulation induced by the endophytic fungus Phomopsis in Betula platyphylla. Treatment of birch plantlets with the endophytic fungus Phomopsis promoted an NO burst and accumulation of PAs and betulin. Birch plantlets were treated with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the PA synthesis inhibitor d-arginine (d-arg). cPTIO and d-arg inhibited the fungus-induced NO burst and accumulation of PAs and betulin. The exogenous NO donor sodium nitroprusside promoted PA production and betulin accumulation, whereas an exogenous PA, putrescine, promoted an NO burst and betulin accumulation. In addition, d-arg inhibited NO production and cPTIO decreased PA production during fungus-induced betulin accumulation. Our results indicate that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.     

Homologous and heterologous desensitization and synergy in pathways leading to the soybean oxidative burst

Because the H₂O₂ and O₂ ⁻ generated during a pathogen-triggered oxidative burst could either protect or destroy a besieged plant cell, their synthesis might be expected to be tightly regulated. We have examined the nature of this regulation as it is communicated between homologous and heterologous oxidative-burst pathways, using both chemical (oligogalacturonic acid, harpin, fensulfothion) and mechanical (osmotic stress) stimuli to induce the burst. We report here that the above three chemical elicitors attenuate a subsequent oxidative burst induced in cultured soybean (Glycine max L.) cells by either the same (homologous desensitization) or a different chemical elicitor (heterologous desensitization). Further, when the magnitude of the initial oxidative burst is maximal, the cells remain refractory to subsequent elicitation for at least 10 min and then revive their sensitivities to re-stimulation with a half-time of >20 min. Mechanical stimulation of the oxidative burst appears to be regulated by a different set of constraints. Although initiation of a mechanically induced burst leads to attenuation of a subsequent mechanically induced burst, the same mechanical stimulus is peculiarly unable to reduce a subsequent chemically induced burst. The converse is also true, suggesting that heterologous desensitization of the oxidative burst does not extend to mixed chemical and mechanical/osmotic stimuli. However, communication between these disparate forms of elicitation is still demonstrated to occur, since low-level chemical stimuli strongly synergize concurrent low-level osmotic stimuli and vice versa. Furthermore, the pattern of synergy changes dramatically if one stimulus is administered immediately prior to the other. Taken together, these data demonstrate that significant cross-talk occurs among the different signaling pathways of the oxidative burst and that the overall process is tightly regulated. Received: 10 January 2000 / Accepted: 22 February 2000     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.     

Studies on the mechanism of Ca2+ stimulation of plasminogen activator synthesis/release by Swiss 3T3 cells.

Stimulation of postconfluent Swiss 3T3 cells in serum-free medium with 4.3 mM Ca2+ results in marked increases in both released and cell-associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post-stimulation and continued to increase steadily until 48 hours at which time the stimulates cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.     

Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.     

Characterization of the Xanthomonas campestris pv. campestris lipopolysaccharide substructures essential for elicitation of an oxidative burst in tobacco cells

The lipopolysaccharides (LPS) of gram-negative bacteria are essential for perception of pathogens by animals and plants. To identify the LPS substructure or substructures recognized by plants, we isolated water-phase (w)LPS from different Xanthomonas campestris pv. campestris mutants and analyzed their sugar content and ability to elicit an oxidative burst in tobacco cell cultures. The different wLPS species are characterized by lacking repetitive subunits of the O-antigen, the complete O-antigen, or even most of the core region. Because loss of lipid A would be lethal to bacteria, pure lipid A was obtained from X. campestris pv. campestris wild-type wLPS by chemical hydrolysis. The elicitation experiments with tobacco cell cultures revealed that LPS detection is dependent on the bioavailability of the amphiphilic wLPS, which can form micelles in an aqueous environment. By adding deoxycholate to prevent micelle formation, all of the tested wLPS species showed elicitation capability, whereas the lipid A alone was not able to trigger an oxidative burst or calcium transients in tobacco cell cultures. These results suggest that the LPS substructure recognized by tobacco cells is localized in the inner core region of the LPS, consisting of glucose, galacturonic acid, and 3-deoxy-d-manno-oct-2-ulosonic acids. Although lipid A alone seems to be insufficient to induce an oxidative burst in tobacco cell cultures, it cannot be ruled out that lipid A or the glucosamine backbone may be important in combination with the inner core structures.