Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. Emerging data indicate that the oxidative burst reflects activation of a membrane-bound NADPH oxidase closely resembling that operating in activated neutrophils. The oxidants are not only direct protective agents, but H2O2 also functions as a substrate for oxidative cross-linking in the cell wall, as a threshold trigger for hypersensitive cell death, and as a diffusible signal for induction of cellular protectant genes in surrounding cells. Activation of the oxidative burst is a central component of a highly amplified and integrated signal system, also involving salicylic acid and perturbations of cytosolic Ca2+, which underlies the expression of disease-resistance mechanisms.

The oxidative burst in plant disease resistance

Plants are constantly being challenged by aspiring pathogens, but disease is rare. Why? Broadly, there are three reasons for pathogen failure. Either (1) the plant is unable to support the niche requirements of a potential pathogen and is thus a non? host; or (2) the plant possesses preformed structural barriers or toxic compounds that confine successful infections to specialized pathogen species; or (3) upon recognition of the attacking pathogen, defense mechanisms are elaborated and the invasion remains localized. All three types of interaction are said to be incompatible, but only the latter resistance mech? anism depends on induced responses. Successful pathogen invasion and disease (compatibility) ensue if the preformed plant defenses are inappropriate, the plant does not detect the pathogen, or the activated defense responses are ineffective. In this review, we examine the essential prerequisites for patho? gen recognition and the induction of localized defense responses. Preformed defenses are considered elsewhere in this issue (see Osbourn, 1996, in this issue). Race-specific pathogen recognition is hypothesized to re? sult from the direct or indirect interaction of the product of a dominant or semidominant plant resistance (R) gene with a product derived from the corresponding dominant pathogen avirulence (Avr) gene (Keen, 1992; Staskawicz et al., 1995). Subsequent signal transduction events are assumed to coordinate the activation of an array of defense responses. This "simple" model appears to explain much but begs many questions. For example, R gene products are likely to provide key components for recognition, but how do the distinct classes of R proteins characterized to date (see Bent, 1996, in this is? sue) activate the defense response? Do different R gene classes activate distinct responses? The regulation of some components of defense mechanisms has been studied in plant cell cultures in response to non-race-specific elicitors, but to what extent do such studies provide a model for R gene func? tion? Plant resistance is often correlated with the activation of specific defense responses, but which (if any) are required to abolish or retard pathogen growth, and how? Which are pri? mary responses and which are secondary? Does the first response involve transcriptional regulation, the activation of preformed enzymes, and/or the opening of ion channels, or

Resistance gene-dependent plant defense responses.

Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

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Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response

Plants adapt to environmental stresses through specific genetic responses. The molecular mechanisms associated with signal transduction, leading to changes in gene expression early in the stress response, are largely unknown. It is clear, however, that gene expression associated with acclimatory responses is sensitive to the redox state of the cell. Of the many components which contribute to the redox balance of the cell, two factors have been shown to be crucial in mediating stress responses. Thiol/disulphide exchange reactions, particularly involving the glutathione pool and the generation of the oxidant H2O2, are central components of signal transduction in both environmental and biotic stresses. These molecules are multifunctional triggers, modulating metabolism and gene expression. Both are able to cross biological membranes and diffuse or be transported long distances from their sites of origin. Glutathione and H2O2 may act alone or in unison, in intracellular and systemic signalling systems, to achieve acclimation and tolerance to biotic and abiotic stresses.

Hydrogen peroxide‐ and glutathione‐associated mechanisms of acclimatory stress tolerance and signalling

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides.

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 appe...

The Origin of the Oxidative Burst in Plants

Plant cytochrome P450.

A cationic (pI 8.3) wall-bound peroxidase has been purified to homogeneity from suspension-cultured cells of French bean (Phaseolus vulgaris L.). The enzyme was a glycoprotein and its M(r) was 46,000 as determined by SDS/Page and h.p.l.c. gel filtration. It was localized biochemically to microsomes and the cell wall, and the latter subcellular distribution was confirmed by immunogold techniques. The native enzyme showed absorption maxima at 403, 500 and 640 nm, with an RZ (A405/A280) of 3.3. The peroxidase oxidized guaiacol and natural phenolic acids. By desorption-chemical-ionization mass spectrometry the enzyme was found to oxidize the model compound, ferulic acid, into dehydrodiferulic acid. Kinetics studies indicated an apparent Km of 113.3 +/- 22.9 microM and a Vmax of 144 mumol.min-1.nmol-1 of protein at an H2O2 concentration of 100 microM. In comparison with a second French-bean peroxidase (FBP) and horseradish peroxidase, as a model, it acted with a 6-10-fold higher specificity in this capacity. It is a member of the peroxidase superfamily of bacterial, fungal and plant haem proteins by virtue of its highly conserved amino acid sequence within the proximal and distal haem-binding sites. This is good evidence that this particular FBP may function in constructing covalent cross-linkages in the wall during development and response to pathogens. Images

Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.).

The purification of glycosyltransferases involved in wall matrix polysaccharide synthesis has been attempted. A number of activities readily demonstrated in isolated Golgi membranes are lost following detergent solubilization. However, solubilization releases pyrophosphorylases and phosphatases that hydrolyse the substrate in enzyme assays, whether UDP-glucose, -arabinose or -xylose is used. This hydrolysis, which cannot be completely inhibited, appears to be the major factor in the apparent loss of activity. Separation of this hydrolytic activity during further purification by ion-exchange and gel exclusion leads to recovery of glycosyltransferase activity. Thus two xylosyltransferases and one arabinosyltransferase could be partially purified. These appeared to be differentially expressed. The arabinosyltransferase of apparent M(r) 70,000 on size-exclusion chromatography was isolated from cells undergoing rapid growth and division. A xylosyltransferase of apparent M(r) 38,000 on size-exclusion chromatography was associated with cell expansion and primary wall synthesis. A second xylosyltransferase, which was purified to near homogeneity with M(r) 40,000, showed a peak of activity during the period of maximum secondary wall synthesis.

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Partial purification of Golgi-bound arabinosyltransferase and two isoforms of xylosyltransferase from French bean (Phaseolus vulgaris L.).

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) purified from suspension-cultured cells of French bean (Phaseolus vulgaris) has been further characterized. A number of techniques, including use of an antiserum and affinity probes, have established that all the antigenic polypeptides represent polymorphic Mr forms of the enzyme. These peptides include an apparently higher-Mr (83,000) form which shows different kinetics of induction from the Mr-77000 forms that have been extensively characterized previously. The larger subunit appeared to be PAL by the following criteria: (a) binding to specific affinity and antibody matrices; (b) peptide mapping; (c) active-site labelling; and (d) amino acid composition. The increased Mr of the larger subunit was not completely attributable to glycosylation, although some sugar residues were detected in this Mr-83000 form but not in the other Mr forms. Mr-83000 subunits were also immunoprecipitated from translations in vitro of mRNA from cells that had been stressed for a long period. They were also detected in leaf tissues that were not yet undergoing an extensive wound response. This form of the enzyme may be constitutive and involved in the low-level accumulation of phenolics in most cell types. By contrast, the Mr-77000 forms of PAL were rapidly induced during elicitor action, wounding or cytokinin-induced xylogenesis as a key regulatory enzyme involved in the synthesis of phenolics under stress conditions or during differentiation.

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G P Bolwell

Papers

The Origin of the Oxidative Burst in Plants

Plant cytochrome P450.

Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.).

Partial purification of Golgi-bound arabinosyltransferase and two isoforms of xylosyltransferase from French bean (Phaseolus vulgaris L.).

L-phenylalanine ammonia-lyase from French bean (Phaseolus vulgaris L.). Characterization and differential expression of antigenic multiple Mr forms.