The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Plasma membrane redox and regulation of cell growth

Summary Gene expression can be activated by external oxidants which are reduced at the cell surface by plasma membrane electron transport. The signals generated in response to the plasma membrane electron transport include activation of proton release, internal calcium changes, and change in reductant/oxidant ratio in the cytosol. H₂O₂ generated in response to ligands which bind to plasma membrane receptors can also activate protein tyrosine kinases and gene expression. Inhibition of oxygen radical generation at the cell surface in response to the mitogen, phorbol myristate acetate by retinoic acid is consistent with a role for the plasma membrane electron transport as the source for H₂O₂ in Balb 3T3 cells. Agents which affect the binding of coenzyme Q to redox sites in the plasma membrane electron transport may increase formation of semiquinone radicals in the membrane which can be a source of oxygen radicals and H₂O₂. The generation of H₂O₂ by transformed cells indicates that oncogene product expression in the plasma membrane may also increase quinone-based oxygen radical generation.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

Optimising hydrogen peroxide measurement in exhaled breath condensate

Abstract

Background: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H₂O₂), have been measured in EBC, without adequate published details of validation and optimisation.

Objectives: To explore factors that affect accurate quantitation of H₂O₂ in EBC.

Materials and Methods: H₂O₂ was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature.

Results: Standard curve slope was significantly altered by pH, leading to a potential difference in H₂O₂ quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H₂O₂ added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H₂O₂ ranged from 3.4 nM to 8.8 nM depending on the buffer used.

Conclusions: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H₂O₂ in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H₂O₂ in EBC requires rigorous optimisation.     

Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures

Two strains of Lactobacillus plantarum accumulated H₂O₂ when grown aerobically in a complex glucose based medium. The H₂O₂ accumulation did not occur immediately on exposure of the culture to O₂ but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O₂ utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H₂O₂ was not a product of NADH oxidase in vitro but was formed in substantial quantities from O₂ during oxidation of pyruvate. The three enzymes were induced by O₂ and H₂O₂; the induction of NADH oxidase responded to lower levels of O₂ (but not of H₂O₂) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source - TPP thiamin pyrophosphate     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Selection of Vaginal H2O2-Generating Lactobacillus Species for Probiotic Use

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H₂O₂. This paper describes the selection of H₂O₂-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H₂O₂ level, was chosen to study: (a) the kinetics of H₂O₂ production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H₂O₂ in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H₂O₂L⁻¹) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H₂O₂. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H₂O₂ since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract. Received: 17 November 1998 / Accepted: 17 December 1998     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

High light‐induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability

The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO₂ availability putatively includes enhanced ROS production in the so‐called Mehler reaction. Such conditions are thought to encourage O₂ to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical () and hydrogen peroxide (H₂O₂). In contrast, here it is shown in Chlamydomonas reinhardtii that CO₂ depletion under high light levels lowered cellular H₂O₂ production, and that elevated CO₂ levels increased H₂O₂ production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H₂O₂ production under high CO₂ availability. High light levels under low CO₂ availability induced photoprotective mechanisms called non‐photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE‐deficient mutant npq4 produced more H₂O₂ than wild‐type cells under high light levels, although less so under high CO₂ availability, whereas it demonstrated equal or greater enzymatic H₂O₂‐degrading capacity. The qT‐deficient mutant stt7‐9 produced the same H₂O₂ as wild‐type cells under high CO₂ availability. Physiological levels of H₂O₂ were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO₂ availability wild‐type cells behaved like stt7‐9 cells stuck in state 1.     

Two sources of endogenous hydrogen peroxide in Escherichia coli

Mechanisms of hydrogen peroxide generation in Escherichia coli were investigated using a strain lacking scavenging enzymes. Surprisingly, the deletion of many abundant flavoenzymes that are known to autoxidize in vitro did not substantially lessen overall H₂O₂ formation. However, H₂O₂ production diminished by 25–30% when NadB turnover was eliminated. The flavin‐dependent desaturating dehydrogenase, NadB uses fumarate as an electron acceptor in anaerobic cells. Experiments showed that aerobic NadB turnover depends upon its oxidation by molecular oxygen, with H₂O₂ as a product. This reaction appears to be mechanistically adventitious. In contrast, most desaturating dehydrogenases are associated with the respiratory chain and deliver electrons to fumarate anaerobically or oxygen aerobically without the formation of toxic by‐products. Presumably, NadB can persist as an H₂O₂‐generating enzyme because its flux is limited. The anaerobic respiratory enzyme fumarate reductase uses a flavoprotein subunit that is homologous to NadB and accordingly forms substantial H₂O₂ upon aeration. This tendency is substantially suppressed by cytochrome oxidase. Thus cytochrome d oxidase, which is prevalent among anaerobes, may diminish intracellular H₂O₂ formation by the anaerobic respiratory chain, whenever these organisms encounter oxygen. These two examples reveal biochemical and physiological arrangements through which evolution has minimized the rate of intracellular oxidant formation.     

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

Aims: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H₂O₂) to inactivate bacteria on and within fresh produce. Methods and Results: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H₂O₂ under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H₂O₂ concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1·5% H₂O₂ at 50°C, UV dose of 37·8 mJ cm^?2), the surface Salmonella were reduced by 4·12 ± 0·45 and internal counts by 2·84 ± 0·34 log CFU, which was significantly higher compared with H₂O₂ or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV–H₂O₂‐treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25°C. Conclusions: UV–H₂O₂ reduce the bacterial populations on and within fresh produce without affecting the shelf‐life stability. Significance of the Study: UV–H₂O₂ represent an alternative to hypochlorite washes to decontaminate fresh produce.     

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract

The production of reactive oxygen species, including hydrogen peroxide (H₂O₂), is increased in diseased blood vessels. Although H₂O₂ leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO₂ (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H₂O₂. The relaxant response to BAY 41-2272 (pD₂: 6.79?±?0.10 and 6.62?±?0.17), BAY 60-2770 (pD₂: 9.57?±?0.06 and 9.34?±?0.15) or 8-Br-cGMP (pD₂: 5.19?±?0.06 and 5.24?±?0.08) was not apparently affected by exposure to H₂O₂. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H₂O₂ was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H₂O₂ (pD₂: 8.73?±?0.05 and 8.30?±?0.05), which was normalized in the presence of catalase (pD₂: 8.59?±?0.05). Likewise, H₂O₂ exposure impaired the relaxant response to acidified NaNO₂ (pD₂: 6.52?±?0.17 and 6.09?±?0.16). These findings suggest that H₂O₂ interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.     

Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination

Laboratory experiments targeting the effect of humic acid (HA) on the cell lysis of cyanobacterium Anabaena circinalis have been performed. Light irradiation was found to be an important factor for the cell lysis phenomenon, whereas intracellular hydrogen peroxide (H₂O₂) might be a chemical factor for the process. An exogenous H₂O₂ concentration of 1.0 mg l⁻¹ was determined as the threshold for cell survival. Our results indicated that HA or its possible product(s) of photochemical reaction can induce damage to intracellular catalase under artificial illumination, which leads intracellular H₂O₂ to be accumulated to an abnormally high concentration, eventually resulting in cell death. Moreover, H₂O₂ released into the culture from dead cells can damage other cells, which in turn brings about the population extinction.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Expression of antioxidant defense genes in mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) roots under water-logging is associated with hypoxia tolerance

This study was conducted to examine the extent of oxidative stress and the role of antioxidant enzymes on hypoxia tolerance in highly tolerant wild species Vigna luteola, and mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (susceptible). Two days of water-logging caused about 40–50% decline in superoxide radical (O₂ ^·−) and hydrogen peroxide (H₂O₂) contents in all the genotypes, however, further water-logging to 8 days caused significant increase in O₂ ^·− and H₂O₂ contents, and the values were 80–90% of the control values. In control and revived plants O₂ ^·− and H₂O₂ contents were higher in Pusa Baisakhi, while under water-logging stress T 44 and V. luteola showed greater increases in the O₂ ^·− and H₂O₂ contents. Hypoxia induced increase in superoxide dismutase, ascorbate peroxidase, and glutathione reductase activities were higher in T 44 and V. luteola compared with Pusa Baisakhi; and the increases in T 44 and V. luteola continued up to 8th day of water-logging, while in case of Pusa Baisakhi, the maximum increase was observed only on the 2nd day of water-logging. Gene expression studies showed enhanced expression of cytosolic-Cu/Zn-superoxide dismutase (SOD) and cytosolic-ascorbate peroxidase (APX) in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of Pusa Baisakhi. PCR band products were cloned and sequenced, and partial cDNAs of Cu/Zn-SOD and APX, respectively, were obtained. Results suggest that increase in the activity of antioxidant enzymes is to scavenge reactive oxygen species produced both during and after relief from water-logging stress.     

Immediate response of Chara braunii exposed to zinc and hydrogen peroxide

The immediate effect of zinc (Zn) and hydrogen peroxide (H₂O₂) in Chara braunii was analyzed in short-time exposure experiments. The exposure concentrations were 12.3, 18.4, and 24.5 μmol L^?1 H₂O₂, 12, 60, and 120 mg L^?1 Zn, and 12.3 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn, 12.3 μmol L^?1 H₂O₂ + 60 mg L^?1 Zn, and 18.4 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn. The stress response of C. braunii was analyzed by measuring photosynthetic photosystem II activity, chlorophyll a and b and carotenoid contents, the H₂O₂ concentration, and antioxidant enzyme activities of ascorbic peroxidase, catalase, and guaiacol peroxidase. The short-term addition of Zn reduced pigment contents in C. braunii. Chlorophyll a and b and carotenoid contents in H₂O₂-exposed C. braunii were as high as in control plants. Photosynthesis was reduced in H₂O₂-treated C. braunii and the short-term addition of Zn did not affect the electron transport rate. H₂O₂ concentration and antioxidant enzyme activities in C. braunii were not significantly different between control and exposed plants. Trends of enzymatic adaptation were described: the H₂O₂-induced stress response was characterized by increased antioxidant enzyme activities, whereas Zn inactivated catalase in C. braunii.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

Hydrogen peroxide accumulation in Medicago truncatula roots colonized by the arbuscular mycorrhiza-forming fungus Glomus intraradices

The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H₂O₂ in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H₂O₂ accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H₂O₂ was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H₂O₂ accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H₂O₂ accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices. Received: 1 October 1998 / Accepted: 22 December 1998