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 extra-cellular 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.

Summary. Wound stress activated wheat root cells to produce oxygen radicals. The production was accompanied by an increased permeability for potassium ions and a depolarization of the plasma membrane. Various electron donors, such as the nonpenetrating donor potassium ferrocyanide as well as NADH and NADPH, caused the amplification of superoxide production by root cells. The Oa'--generating system in wheat root cells was found to be considerably sensitive to diphenylene iodonium, which is generally considered as a suicide inhibitor of neutrophil NADPH oxidase, and to other inhibitors of flavoprotein activity, chlorpromazine and quinine. The xenobiotic compound amidopyrine caused activation of the 02--generating system, which was depressed by DPI. The O2'--generating system in root ceils was shown to be significantly dependent on calcium content. Calcium loading of the root cells induced a powerful increase of the superoxide release. Data obtained indicate that superoxide generation is one of the early events of the wound stress response. Redox systems of the plasma membrane may be involved in the superoxide production in response to wound stress and detoxification of xenobiotic compounds in root ceils.

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The superoxide generation in the plant root cells in response to wound stress has been studied by the electron spin resonance (ESR) spin-trap and epinephrine-adrenochrome acceptor methods. Tiron readily oxidized by Oz -to a rather stable free radical semiquinone was used as a spin trap. Wound stress was shown to activate the root cells inducing an increase in superoxide production. The largest amount of superoxide was registered in the early stage after excision of the roots from the seedlings (over 1-2 h). Further incubation of the roots for 5 and 6 h resulted in the lowering of the superoxide level. Electron donors NADH and NADPH, nonpenetrating via plasma membrane, caused the amplification of superoxide production in root cells, whereas oxidized nucleotide NAD did not affect the Oz-synthesis. Treatment of the roots with a water-soluble analog of naphthoquinone, vitamin K3, led to the total disappearance of the ESR signal from Tiron semiquinone and suppression of epinephrine-adrenochrome conversion. An excessive amount of calcium ions in the root cells induced a powerful increase in the superoxide release and disturbed the adaptation. The data obtained give us a further indication that the redox system of plasma membrane, comprising a flavoprotein, is likely involved in the production of superoxide occurring in the response to wound stress in root cells.

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