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     

Characterization of the Oligogalacturonide-Induced Oxidative Burst in Cultured Soybean (Glycine max) Cells

The rapid release of H2O2 by elicited plant cells, recently termed the oxidative burst, was investigated in suspension-cultured soybean (Glycine max Merr. cv Kent) cells stimulated with a purified polygalacturonic acid (PGA) elicitor. Examination of the elicited cells by fluorescence microscopy revealed that virtually every living cell participates in the elicitor-induced H2O2 burst. Measurement of the kinetics of the response using a macroscopic fluorescence-based assay indicated that approximately 100 molecules of H2O2 are generated per PGA molecule added, achieving a cumulative H2O2 concentration of approximately 1.2 mmol L-1 of packed cells. At the height of the defense response, 3 x 10-14 mol of H2O2 cell-1 min-1 are produced, a value comparable to the rate of H2O2 production by myeloid cells of mammals. Variables affecting the rate and magnitude of the soybean oxidative burst were found to be mechanical stress, extracellular pH, and cell age. The PGA-induced oxidative burst was shown to undergo both homologous and heterologous desensitization, a characteristic of signal transduction pathways in animals. Homologous desensitization was obtained with PGA, and heterologous desensitization was observed with the G protein activator mastoparan, consistent with earlier observations showing that G proteins perform a regulatory function in this pathway. Finally, a model describing the possible role of the PGA-induced oxidative burst in the overall scheme of plant defense is proposed.     

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.     

Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species.

A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed. We have used a dominant-acting calmodulin mutant (VU-3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co-enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures. VU-3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post-translationally, and as a result it hyperactivates calmodulin-dependent NAD kinase. Cells expressing VU-3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli. Increases in NADPH level were also greater in VU-3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst. These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis.     

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca²⁺ channels. Inhibition of the oxidative response impaired Cl⁻ efflux, K⁺ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Cytosolic Ca2+ pulses and protein kinase activation in the signal transduction pathways leading to the plant oxidative burst

The oxidative burst, the rapid production of O₂- and H₂O₂ by plant cells in response to pathogens and Stressors, is a critical step in plant disease resistance and is controlled by several different elicitor-initiated signaling pathways. While different defense elicitors appear to activate disparate initial steps in signaling the oxidative burst, all of the elicitors tested thus far appear to stimulate pathways that converge on the same three core signaling intermediates: 1) the Ca₂₊-independent activation of a mitogen-activated protein kinase (MAPK) family member, 2) the influx of Ca₂₊ into the cytosol, deriving most critically from an internal compartment, and 3) the Ca²⁺-dependent activation of additional protein kinases including a second MAPK homologue and possibly calcium dependent protein kinases (CDPKs). Data from several recent reports are summarized to place these signaling events into a complete and updated model of signaling to the plant oxidative burst.     

Sensing of osmotic pressure changes in tomato cells

Cells of tomato (Lycopersicon esculentum) growing in suspension gradually depleted their culture medium and caused a steady decrease in its osmolality. When confronted with a sudden change in medium osmolality (a hypo-osmotic or hyperosmotic shock), respectively, these cells responded with volume changes and stress symptoms such as rapid extracellular alkalinization, efflux of K(+)-ions, and induction of 1-aminocyclopropane-1-carboxylate synthase acid, the key enzyme of ethylene biosynthesis. This array of stress symptoms is well known from cultured plant cells treated with microbial elicitors. Compared with elicitor treatment, induction of responses by hyperosmotic shock was slow and occurred only after increases of approximately 200,000 Pa in osmotic pressure. In contrast, hypo-osmotic shock induced responses without measurable lag and faster than elicitor treatments. Measurable medium alkalinization was induced when medium osmolality was reduced by as little as approximately 10 mosmol, a change corresponding to only approximately 0.2 bar in osmotic pressure. Like treatment with elicitors, hypo-osmotic shock induced specific changes in protein phosphorylations as demonstrated by in vivo labeling with [(33)P]orthophosphate. Exposure of cells to consecutive up- and down-shifts in medium osmolality showed that sensing of osmotic changes occurred within seconds, whereas adaptation to new osmotic conditions proceeded over hours. In conclusion, suspension-cultured plant cells display rapid, easily measurable macroscopic responses to osmotic shock and provide an interesting model system to study osmoregulation, a key process in plant growth and development.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong?type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.     

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.     

Physiological and genetic responses of bacteria to osmotic stress.

The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary response of bacteria to exposure to a high osmotic environment is the accumulation of certain solutes, K+, glutamate, trehalose, proline, and glycinebetaine, at concentrations that are proportional to the osmolarity of the medium. The supposed function of these solutes is to maintain the osmolarity of the cytoplasm at a value greater than the osmolarity of the medium and thus provide turgor pressure within the cells. Accumulation of these metabolites is accomplished by de novo synthesis or by uptake from the medium. Production of proteins that mediate accumulation or uptake of these metabolites is under osmotic control. This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.     

Oligocarrageenans and tissue-dependant oxidative burst in Solieria chordalis (Rhodophyceae, Gigartinales)

The release of hydrogen peroxide by thallus fragments of the rhodophycean Solieria chordalis (C. Agardh) J. Agardh has been documented both in the presence and in the absence of oligosaccharides. Within 1 h, ramuli were able to release large amounts of peroxide in the absence of any chemical stress. Among potential elicitors tested, only degree of polymerization 1 (DP1) and DP7‐8 oligo‐iota‐carrageenans stimulated defense mechanisms in both axes and ramuli as shown by the occurrence of an oxidative burst. Chopping of the tissues had no effect on the intensity of the burst, therefore suggesting that mainly cortical cell layers were involved in the process. After 5 min incubation, a dose of 125 μg mL^?1 of an oligomeric mixture containing a large proportion of DP1 units proved to be sufficient to obtain a maximal response. The intensity of the burst was significantly higher with isolated ramuli than with pieces of the axis, with outer peroxide accumulations reaching 200 nmol g^?1 fresh weight of treated tissue. Altogether, our results show that S. chordalis is able to react to a simulated pathogen attack by an oxidative burst and that the capacity to carry out an oxidative burst is stronger in ramuli than in axes.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Rapid Stimulation of an Oxidative Burst during Elicitation of Cultured Plant Cells : Role in Defense and Signal Transduction

Stimulation of cultured plant cells with elicitors of the defense response leads to the rapid destruction of a variety of water-soluble compounds including indoleacetic acid and certain fluorescent dyes. This destructive activity, which is often vigorously manifested within 5 minutes of elicitor addition, is shown to derive from the rapid production of H₂O₂ and its use by extracellular peroxidases. Because of its speed of appearance, this oxidative burst may qualify as the first induced line of defense against invading pathogens. Since H₂O₂ has been implicated as a second messenger of hormone-stimulated metabolic changes in some animal cells, its possible role in transduction of the defense signal in plants was also examined. Not only did exogenous H₂O₂ alone stimulate phytoalexin production in the plant cell suspension, but inhibition of elicitor-stimulated phytoalexin production was observed upon addition of catalase and other inhibitors of the oxidative burst. Furthermore, for inhibition to occur, the presence of catalase was required during elicitor addition, since if introduction of the enzyme was delayed until 1 hour after addition of the elicitor, no inhibition resulted. These results suggest that H₂O₂ also plays an important role in inducing subsequent defense responses such as phytoalexin production.     

Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks.

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.     

The oxidative burst in plant defense: Function and signal transduction

The rapid production and accumulation of active oxygen species (AOS), the oxidative burst, has been shown to occur in a variety of plant/pathogen systems. In particular, two species, hydrogen peroxide (H₂O₂) and the superoxide radical anion O₂^? have received considerable attention. H₂O₂ and O₂^?, while acting directly as antimicrobial agents, may also serve as second messengers or catalysts in plants to activate a more diverse set of defense responses. Some of the better studied downstream responses promoted by AOS are (1) the cross-linking of cell wall proteins, (2) the induction of defense-related genes, (3) the stimulation of phytoalexin biosynthesis and (4) promotion of the hypersensitive response (HR). A useful model for studying the oxidative burst in plants is the neutrophil NADPH ox-idase complex, the primary source of AOS production in mammals. Several of the subunits of the neutrophil NADPH oxidase complex have been immunologically identified in plants. Furthermore, many of the components known to be involved in the signal transduction pathway in neutrophils have also been found to play a role in the oxidative burst in plants. Just as various ligands activate the oxidase complex in neutrophils, several ligands (elicitors or pathogens) also lead to induction of the oxidative burst in plant cells. The similarities between the neutrophil and plant oxidative bursts will be elaborated in this review. Following stimulation with elicitors, different signal transduction pathways are activated in plants, depending on the source of elicitor used. While the identities and chronologies of the major intermediates in these pathways remain largely unknown, there is strong evidence at least for participation of phospholipases, H⁺/K⁺ exchange, Ca²⁺ influxes, protein kinases and phosphatases, and GTP binding proteins. In an effort to integrate these various signaling events into a single scheme, we have constructed a hypothetical model that proposes how different elicitors might induce the oxidative burst in the same cell by different pathways.     

Cytochalasin E-induced oxidative metabolism in polymorphonuclear leukocytes.

The extent of the cyanide-resistent oxidative burst of polymorpho-nuclear leukocytes after stimulation with cytochalasin E was shown to depend markedly on the osmolarity of the cell-suspension medium. With granulocyte concentrations up to 2 X 10(6) cells/ml, optimal oxygen consumption and releases of H2O2 and superoxide anions were reached at 180 mOsmol and 2 X 10(-5) M cytochalasin E. After removal of unbound activator, the cellular oxidative activity remained unaltered and continued to depend on the used osmotic conditions. It is proposed that binding of cytochalasin to the plasma membrane induces an irreversible activation of the oxidative system, whereas the resulting metabolic activity depends on conformational changes in the plasma membrane.     

Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress

Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.