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