Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary.  Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H⁺ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H⁺ consumption for O₂ ^•− dismutation to H₂O₂. Also K⁺ influx was strongly depressed by MeJA, even transitorily reverting to K⁺ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H₂O₂ accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O₂ uptake by roots gave similar results. These and other results for additions of H₂O₂ or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H₂O₂ being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN⁻- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O₂ ^•− and H₂O₂, O₂ uptake, and peroxidase activity by roots. Received July 12, 2002; accepted October 2, 2002; published online May 21, 2003 RID="*"     

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2) (.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.     

Oxidative burst in suspension culture of Taxus cuspidata induced by a laminar shear stress in short-term

Generation of active oxidative species induced by shear stress in suspension cultures of Taxus cuspidata was investigated in a Couette-type shear reactor. It was found that T. cuspidata cells respond to a shear rate of 95 s(-)(1) with oxidative bursts. Their triphasic characteristics in 6 h were similar in both intracellular H(2)O(2) production and extracellular O(2)(-)( )(*) production. Additionally, inhibition studies with diphenylene iodonium and azide suggested that the key enzyme responsible for oxidative bursts under the shear rate of 95 s(-)(1) is primarily NADPH oxidase and the contribution of peroxidase for oxidative bursts was less. Investigation of the relationship between active oxidative species and defense responses induced by the shear stress indicated that the O(2)(-)( )(*) burst may account for the change of membrane permeability, and the H(2)O(2) burst plays an important role in inducing secondary metabolites such as the activation of phenylalanine ammonia lyase enzyme and phenolic accumulation. Furthermore, oxidative bursts elicited by the shear rate of 95 s(-)(1) were suppressed by treatment with suramin, nifedipine, and neomycin prior to the shear stress treatment, suggesting that G-protein, Ca(2+) channel, and phospholipase C are involved in the signal pathway for oxidative bursts induced by the shear stress. A model is proposed to explain the oxidative burst in cultured T. cuspidata cells challenged with the shear stress.     

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.     

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.     

Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes

The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O(2)(*-)) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O(2)(*-) production. It was found that a muscarinic agonist oxotremorine (OXO; 80 microM) increased O(2)(*-) levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (50 microM) or apocynin (100 microM), these increases in extra- and intracellular O(2)(*-) levels were substantially abolished or attenuated. The O(2)(*-) increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H(2)HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O(2)(*-) was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O(2)(*-) levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O(2)(*-) accumulation. These results suggest that Nox1 represents a major NAD(P)H oxidase isoform responsible for extracellular O(2)(*-) production. This rapid extracellular production of O(2)(*-) seems to be unique to OXO-induced M(1)-receptor activation, since ANG II-induced intra- and extracellular O(2)(*-) increases in parallel. It is concluded that the outward production of O(2)(*-) via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.     

在耐热性诱导中豌豆叶片过氧化氢和水杨酸含量与质膜ATP酶活性的变化及相互关系

在高温锻炼(37℃,2h)过程中,豌豆(Pisum sativum L.)叶片过氧化氢(H_2O_2)和游离态水杨酸(SA)含量与质膜ATP酶(H~ -ATPase)活性都有一个高峰,H_2O_2的迸发早于游离态SA的积累,而质膜H~ -ATPase活性高峰的出现则迟于SA高峰;活性氧清除剂、抗氧化剂、质膜NADPH氧化酶抑制剂和H_2O_2的淬灭剂预处理均可有效地阻止高温下H_2O_2和SA的积累以及质膜H~ -ATPase活性的增加。根据以上结果推测,H_2O_2、质膜H~ -ATPase和SA均参与耐热性诱导相关的信号传递,前者作用于SA的上游,而后者在SA下游起作用。     

Rapid determination of seed vigor based on the level of superoxide generation during early imbibition.

It has been reported that a large amount of reactive oxygen species (ROS) is produced during seed imbibition and this ROS is related to seed vigor. To make this physiological mechanism clear, we have used 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-alpha)pyrazin-3-one (MCLA) as a sensitive and physiologically compatible probe for the determination of superoxide anion (O(2)(*-)) production in vivo. Our results showed that dry rice (Oryzae sativa L.) seed embryo cells possessed the capacity to generate O(2)(*-). Conversely, the O(2)(*-) production of seed embryo cells was inhibited by quinacrine (QA) and diphenylene iodonium (DPI), two specific inhibitors of NADPH oxidase, and O(2)(*-) induced MCLA-mediated chemiluminescence was also blocked by superoxide dismutase (SOD). Additionally, O(2)(*-) -production ability increased dramatically in a NADPH-dependent way in the plasma membrane protein abstract from rice seed embryo cells, whereas SOD and the inhibitors mentioned above suppressed O(2)(*-) production. These preliminary results suggested that rice seeds contained intrinsic NADPH oxidase activity. To validate this conclusion, dichlorofluorescein (DCF) fluorescence staining was used (observed under a laser scanning microscope, LSM) to reflect the in situ assessment of O(2)(*-) -generation. The position of O(2)(*-) production located at the plasma membrane. Additionally the ability to synthesize O(2)(*-) was activated directly by calcium ions. These observations are in accord with the character of NADPH oxidase catalyzed O(2)(*-) -generation. All these results indicated that NADPH oxidase contribute to O(2)(*-) production and release to the outside. We concluded that NADPH oxidase plays an intrinsic role as an NADPH sensor, so, measuring the O(2)(*-) one can monitor the NADPH concentration, which is an index of seed vigor. Therefore the O(2)(*-) generation during early imbibition can serve as a rapid measurement of seed vigor.     

The Generation of Active Oxygen Species Differs in Tobacco and Grapevine Mesophyll Protoplasts

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.     

Exogenous cadaverine induces oxidative burst and reduces cadaverine conjugate content in the common ice plant

The effect of free cadaverine (Cad) on its conjugates formation was analyzed in roots of the common ice plants (Mesembryanthemum crystallinum L.). It was found for the first time that Cad could induce oxidative burst in the roots of adult plants, as was evident from the sharp decrease in the content of Cad soluble or insoluble conjugates. This unusual effect was associated with the increased oxidative degradation of exogenous Cad (1mM, 1.5h) and intense H(2)O(2) production in the root cells of adult plants. Root treatment of both juvenile and adult plants with H(2)O(2) (1mM, 1.5h) reduced the content of soluble Cad conjugates and increased the content of their components, free Cad and phenols. We also found that one of the possible reasons of the negative effect of exogenous diamine on the formation of conjugated forms in adult roots was alkalization of the root apoplast at Cad addition to nutrient medium and the unusual O(2)(-) synthase function as a pH-dependent guaiacol peroxidase in the presence of a high content of H(2)O(2). This was confirmed by the data on the accumulation of O(2)(-) and enhanced superoxide dismutase activity in adult roots under treatment with Cad. It is possible that the accumulation of O(2)(-) together with H(2)O(2) was also responsible for oxidative burst, which induced a decrease in the content of Cad conjugates in adult roots of the common ice plants.     

Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Early events in the perception of lipopolysaccharides in the brown alga Laminaria digitata include an oxidative burst and activation of fatty acid oxidation cascades

This study provides evidence that bacterial lipopolysaccharides can be strong triggers of early events of defence reactions in the brown algal kelp Laminaria digitata, constituting the first report of a biological activity of this class of macromolecules in a marine alga. The early events include an oxidative burst, release of free saturated and unsaturated fatty acids (FFAs) and accumulation of oxylipins such as 13-hydroxyoctadecatrienoic acid and 15-hydroxyeicosapentaenoic acid. The formation of reactive oxygen species can be inhibited by diphenylene iodonium, suggesting that the source is an NAD(P)H oxidase and is similar to the oxidative burst in neutrophils and terrestrial plants. In addition and besides triggering an oxidative burst, the hypolipidemic drug clofibrate also induces the release of FFAs, to a lesser extent than lipopolysaccharides, but it does not induce oxylipin production. Other strong inducers of the oxidative burst in Laminaria such as oligoguluronates could not induce the release of FFAs nor oxylipin production. These results suggest that different signalling pathways are involved in the induction of the oxidative burst and oxylipin production.     

The reactive oxygen species are involved in resistance responses of wheat to the Russian wheat aphid

The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the hydrogen peroxide (H(2)O(2)) content and NADPH oxidase (EC 1.6.3.1) activity was studied in the resistant (cv. Tugela DN) and near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). The objective of this study was to investigate the involvement of the reactive oxygen species (ROS) during the resistance responses against the RWA. Infestation significantly induced an early accumulation of the H(2)O(2) and increase of NADPH oxidase activity to higher levels in the resistant than susceptible plants. Results of inhibitory studies using diphenylene iodonium (DPI), a suicide inhibitor of NADPH oxidase, strongly suggested a possible signalling role for H(2)O(2) during RWA resistance response by activation of downstream defence enzymes [intercellular peroxidase (EC 1.11.1.7) and beta-1,3-glucanase (EC 3.2.1.39)].     

Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng

The mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway responsible for the transduction of signals from the cell surface to the cell interior and the nucleus. MAPKs are involved in vari-ety of physiological process including cell growth, development, meiosis, cell death and cell differentia-tion[1—3]. Typically, the components of MAPK cas-cades include the MAPK, a mitogen-activated protein kinase kinase (MAPKK) and a mitogen-activated pro-tein kinase kinase kin…     

Critical role of an endogenous gastric peroxidase in controlling oxidative damage in H. pylori-mediated and nonmediated gastric ulcer

The objective of the present study is to delineate the mechanism of oxidative damage in human gastric ulcerated mucosa despite the presence of some antioxidant enzymes. We report for the first time the critical role of an endogenous peroxidase, a major H(2)O(2) metabolizing enzyme, in controlling oxidative damage in gastric mucosa. Human gastric mucosa contains a highly active peroxidase in addition to the myeloperoxidase contributed by neutrophil. In both non-Helicobacter pylori (H. pylori)- and H. pylori-mediated gastric ulcer, when myeloperoxidase level increases due to neutrophil accumulation, gastric peroxidase (GPO) level decreases significantly. Moreover, gastric ulcer is associated with oxidative damage of the mucosa as evidenced by significant increase in lipid peroxidation, protein oxidation, and thiol depletion indicating accumulation of reactive oxygen metabolites (ROM). Mucosal total superoxide dismutase (Mn and Cu-Zn SOD) level also decreases significantly leading to increased accumulation of O(2)(*-). To investigate the plausible ROM-mediated inactivation of the GPO during ulceration, the enzyme was partially purified from the mucosa. When exposed to an in vitro ROM generating system, using Cu(2+), ascorbate, and H(2)O(2,) the enzyme gets inactivated, which is dependent on Cu(2+), ascorbate, or H(2)O(2). Insensitivity to SOD excludes inactivation by O(2)(*-). However, complete protection by catalase indicates that H(2)O(2) is essential for inactivation. Sensitivity to EDTA and hydroxyl radical *OH) scavengers indicates that GPO is inactivated most probably by *OH generated from H(2)O(2). We propose that GPO is inactivated in vivo by ROM generated by activated neutrophil. This leads to further accumulation of endogenous H(2)O(2) to cause more oxidative damage to aggravate the ulcer.     

Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae

Rates of H(2)O(2) production by tobacco suspension cells inoculated with zoospores from compatible or incompatible races of the pathogen Phytophthora nicotianae were followed by direct measurement of oxygen evolution from culture supernatants following catalase addition. Rates of HO(2)(*)/O(2)(-) production were compared by following the formation of the formazan of sodium, 3'-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate. In the incompatible interaction only, both reactive oxygen species (ROS) were produced by the cultured host cells in a minor burst between 0 and 2 h and then in a major burst between 8 and 12 h after inoculation. Absolute levels of H(2)O(2) could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H(2)O(2) being formed via dismutation of HO(2)(*)/O(2)(-). The effects of inhibitors of endogenous Cu/Zn superoxide dismutase (diethyldithiocarbamate) and catalase (3-amino-1,2,4-triazole and salicylic acid) were also examined. Yields of ROS in the presence of the inhibitors diphenylene iodonium, allopurinol, and salicylhydroxamic acid suggest that ROS were generated in incompatible host responses by more than one mechanism.     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin

In Arabidopsis thaliana cells, fusicoccin (FC) treatment induced an early and marked increase in the extracellular H(2)O(2) level. It also increased the huge hypo-osmotic stress-induced oxidative wave and, in addition, prevented the H(2)O(2) peak drop. These effects were apparently not linked to changes in either cytoplasmic pH or cytoplasmic free calcium concentration, since they occurred independently of the activity state of the plasma membrane (PM) H(+)-ATPase and neither influx nor efflux of (45)Ca(2+) was modified by FC. In the presence of diphenylene iodonium (DPI), inhibiting the PM NADPH oxidase presumably responsible for reactive oxygen species (ROS) production, no apoplastic H(2)O(2) development was detected either with or without FC. However, no increase in DPI-sensitive ferricyanide reduction, but rather a gradual decrease, occurred with FC. These results suggested that the H(2)O(2) increase observed with FC was not due to a overproduction of ROS but, more probably, to a reduced capability of FC-treated cells to degrade the H(2)O(2) formed. This view, at first supported by the finding that FC-treated cells failed to break down exogenously supplied H(2)O(2), was clearly confirmed by a series of measurements on exogenous catalase activity, tested in cell-free media of FC-treated samples. This assay, in fact, allowed ascertainment and partial characterization of an as yet unidentified factor increasingly accumulating in the incubation medium of FC-treated cells, behaving as a non-competitive catalase inhibitor and able to reduce markedly the cell's capability for H(2)O(2) scavenging.