The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.     

Production of reactive oxygen species in Arabidopsis thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum: implications for basal resistance

The present understanding of ROS generation in the defence responseof Arabidopsis thaliana is reviewed. Evidence suggests thatthe apoplastic oxidative burst generated during basal resistanceis peroxidase-dependent. The ROS generated during this basalresistance may serve to activate NADPH oxidase during the R-gene-mediatedhypersensitive response. The processes involved in the productionof reactive oxygen species in A. thaliana cell suspension culturesin response to an elicitor from Fusarium oxysporum are investigatedin the present work. This system appears analogous to the productionof ROS during the basal resistance response in French bean,which is peroxidase-dependent. A panel of modulators effectivein other pathogen elicitor and plant cell systems has been usedto investigate the Arabidopsis signalling pathways and the plantcell responses involved. Thus as in other systems, an earlycalcium influx into the cytosolic compartment, a rapid effluxof K⁺ and Cl^–, and extracellular alkalinization of elicitedcell cultures has been found. However the alkalinization isnot sufficient to stimulate the apoplastic oxidative burst byitself, unlike in French bean, although vectorial ion fluxesare needed. A secretory component which is sensitive to monensinand N-ethylmaleimide and insensitive to brefeldin A may alsobe necessary for the release and provision of substrates forperoxidase-dependent generation of H₂O₂. Key words: Arabidopsis thaliana, calcium, elicitation, hydrogen peroxide, oxidative burst, secretion     

Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family

Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean. The most important of these, designated FBP1, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor. Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA. Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst. However, FBP1 when expressed in Pichia pastoris generated H₂O₂ using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells. FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells. They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots. This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods. Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.     

Involvement of a class III peroxidase and the mitochondrial protein TSPO in oxidative burst upon treatment of moss plants with a fungal elicitor

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.     

Pathogenic infection and the oxidative defences in plant apoplast

Summary The structural and functional continuum of the plant apoplast is the first site of contact with a pathogen and plays a crucial role in initiation and coordination of many defence responses. In this paper, we present an overview of the involvement of the plant apoplast in plant-pathogen interactions. The process of infection of French bean (Phaseolus vulgaris L.) plants byColletotrichum lindemuthianum is analysed. The ultrastructural features of plant defence responses to fungal infection are then compared with those observed in plants or cell suspensions treated with various elicitors. Changes in cell walls and in whole plant cells responding to infection seem to be highly similar in all systems used. Model systems of French bean and white lupin (Lupinus albus L.) are then utilised to provide some biochemical characteristics of oxidative reactions in the apoplast evoked by elicitor treatment. The species specificity of various mechanisms generating reactive oxygen species is discussed, and some details of pH-dependent H₂O₂-generating activity of peroxidases are demonstrated. As its exocellular nature is an important feature of the oxidative burst, the major consequence of this event, i.e., the oxidative cross-linking of wall components during the papilla formation and strengthening of the walls, is analysed. Finally, the possible involvement of other wall-associated and developmentally regulated H₂O₂-generating mechanisms, like amine and oxalate oxidases, in plant defence is demonstrated. It is concluded that under stress conditions, such apoplastic mechanisms might be employed to increase plants' chances of survival.Abbreviations HR hypersensitive response - IWF intercellular washing fluid - OxO oxalate oxidase - ROS reactive oxygen species - YE elicitor preparation from yeast cell walls     

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris

Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.     

Reactive oxygen species and their role in plant defence and cell wall metabolism

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.     

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H₂O₂. It has been hypothesized that in rose cells H₂O₂ is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H₂O₂ is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H₂O₂ than when treated without the washing procedure. In contrast, a washing treatment reduced the H₂O₂ accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H₂O₂ but did not have a peroxidase capable of forming H₂O₂ in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.     

Effect of auxins and ectomycorrhizal elicitors on wall-bound proteins and enzymes of spruce [Picea abies (L.) Karst.] cells

Summary Elicitors of the ectomycorrhizal fungus Hebeloma crustuliniforme and auxins (IAA, NAA and 2,4-D) were tested for their effects on apoplastic proteins and enzymes of suspension cultured cells of Picea abies (L.) Karst. The ectomycorrhizal elicitor increased the amount of some ionically wall-bound proteins (36, 28, 24, 21 kDa) and decreased the amount of others (61, 22 kDa). The elicitor triggered an H₂O₂ burst and enhanced the peroxidase (EC 1.11.1.7) activity of the Picea cells by increasing one of the two wall-bound peroxidase isoforms. Auxins significantly suppressed the elicitor induction of peroxidase but did not influence the elicitor-triggered H₂O₂ burst. The elicitors and auxin did not change the amount and the pattern of wall-bound invertase isoforms (EC 3.2.1.26) of spruce cells. However, auxin reduced the uptake of glucose by spruce cells and increased the acidification of the cell culture medium. Since Hebeloma lacks apoplastic invertase as well as a sucrose uptake system, utilization of plant-derived sucrose depends on the apoplastic plant invertase activity. Although the host invertase is constitutive, the fungus might be able to increase this invertase activity within a mycorrhiza by lowering the pH of the interface towards the pH optimum of the enzyme via the action of auxin. This fungus-released hormone could increase the H⁺ extrusion of plant cells by activation of the plant membrane H⁺-ATPases. Additionally, an auxin-dependent suppression of glucose uptake by cortical root cells could improve the glucose supply for the fungus. Furthermore, the fungal auxin might suppress the elicitor induced formation of defense enzymes, such as peroxidase.     

Regulation of CDPKs,including identification of PAL kinase,in biotically stressed cells of French bean

Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an M _r of 55000 on the basis of gel migration, ⁴⁵Ca²⁺ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar M _r showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.     

The Origin of the Oxidative Burst in Plants

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the “oxidative burst” of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H₂O₂ appears to be a transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H₂O₂ under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M_r 46,000 wall-bound cationic peroxidase, is able to sustain H₂O₂ production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H₂O₂ from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmaiemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H₂O₂ which does not require such multiple proteinaceous components.     

Redox responses of Arabidopsis thaliana to the green peach aphid,Myzus persicae

The green peach aphid (Myzus persicae) is a phloem-feeding insect that causes economic damage on a wide array of crops. Using a luminol-based assay, a superoxide-responsive reporter gene (Zat12::luciferase), and a probe specific to hydrogen peroxide (HyPer), we demonstrated that this aphid induces accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. Similar to the apoplastic oxidative burst induced by pathogens, this response to aphids was rapid and transient, with two peaks occurring within 1 and 4 hr after infestation. Aphid infestation also induced an oxidative response in the cytosol and peroxisomes, as measured using a redox-sensitive variant of green fluorescent protein (roGFP2). This intracellular response began within minutes of infestation but persisted 20 hr or more after inoculation, and the response of the peroxisomes appeared stronger than the response in the cytosol. Our results suggest that the oxidative response to aphids involves both apoplastic and intracellular sources of ROS, including ROS generation in the peroxisomes, and these different sources of ROS may potentially differ in their impacts on host suitability for aphids.     

The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca²⁺) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca²⁺ burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca²⁺-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca²⁺ burst, supporting its role as a negative regulator of BIK1.     

Chitosan signaling in guard cells requires endogenous salicylic acid

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.     

Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae

The metabolic and cellular changes in source leaves of Nicotiana tabacum L. cv SNN during an incompatible interaction with Phytophthora nicotianae van Breda de Haan were investigated and compared with defence reactions. Hypersensitive cell death was preceded by a rapid and highly localized shift to non-assimilatoric metabolism. During the first 6 h post infection (hpi), reactive oxygen species (ROS) accumulated. Callose was deposited at the interface of adjacent mesophyll cells (≥1 hpi), the export of sucrose collapsed and its content in the apoplast increased. Stomata closed and photosynthetic flux was reallocated from CO₂ assimilation in favour of photorespiration. This was accompanied by an increase in respiration, glucose-6-phosphate dehydrogenase (G6PDH) activity, apoplastic invertase and hexose content. Later (>6 hpi) the photosynthetic electron transport chain was interrupted and photosynthesis completely collapsed. This was accompanied by a further increase in apoplastic invertase and carbohydrates, respiration and oxidative pentose phosphate pathway (OPPP) and followed by further burst in ROS release. Hypersensitive cell death did not appear until photosynthesis completely declined. Photosynthesis was visualized by chlorophyll-a fluorescence imaging on a macro- and microscopic scale. Decline in photosynthesis and defence reactions were highly localized processes, which occur in single mesophyll cells. We propose that in photoautotrophic leaves, photosynthesis and assimilatory metabolism must be switched off to initiate respiration and other processes required for defence. An early blockage of intercellular sugar transportation, due to callose deposition, in conjunction with enhanced apoplastic invertase activity could facilitate this metabolic shift.     

Superoxide‐producing NAD(P)H oxidases in plasma membrane vesicles from elicitor responsive bean plants

Higher plants produce active oxygen species (AOS) that regulate their defence responses against pathogenic elicitation. Etiolated bean seedlings ( Phaseolus vulgaris L. cv. Limburgse vroege) were used to measure the in vivo‐induced AOS production and to search for plasma membrane bound NAD(P)H‐dependent oxidases producing AOS. Immersed bean plants showed a substantial production of H₂O₂, as determined by the peroxidase (EC 1.11.1.7)‐dependent oxidation of 3,5‐dichloro‐2‐hydroxybenzenesulfonic acid (DHBS). Addition of the elicitor polygalacturonase (PGase, EC 3.2.1.15) from Aspergillus japonicus or the phosphatase inhibitor, cantharidin, resulted in a transient increase of AOS synthesis. Plasma membrane vesicles, purified from etiolated bean seedlings, showed an NAD(P)H‐dependent superoxide (O₂⁻) production that was highly stimulated with naphthoquinones. Protein solubilisation and anion exchange chromatography resolved a basal and three naphthoquinone‐stimulated NAD(P)H‐dependent O₂⁻ oxidase fractions. The natural phenol, apigenin, was also a strong inducer of the naphthoquinone‐dependent enzymes, when it was used in the presence of peroxidase. Although, the relation of these different in vitro‐determined plasma membrane NAD(P)H‐dependent O₂⁻ oxidases to the in vivo elicitation of H ₂O₂ has not been elucidated so far.