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Apoplastic Peroxidase Generates Superoxide Anions in Cells of Cotton Cotyledons Undergoing the Hypersensitive Reaction to Xanthomonas campestris pv. malvacearum Race 18

Cotton cotyledons displayed a hypersensitive reaction (HR) in the resistant cultivar Reba B50 after infiltration with the avirulent race 18 of Xanthomonas campestris pv. malvacearum (Xcm). Generation of active oxygen species during the HR was studied biochemically and cytochemically. O₂·¯ was detected in cotyledon disks by the cytochrome c reduction assay 3 h after inoculation. This activity was inhibited by superoxide dismutase (SOD) and by the peroxidase inhibitors salicylhydroxamic acid (SHAM) and KCN but not by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Strong NADH oxidation activity also was found 3 h after inoculation in crude extracts or in apoplastic washing fluid and was dramatically decreased after treatment with SHAM or KCN. NADH oxidation was activated by 2,4- dichlorophenol and MnCl₂, indicating the involvement of a peroxidase. Activity of cationic peroxidase isoforms (pI 9 to 9.5) constitutively expressed in cotyledons was found to be enhanced 3 h after inoculation in the resistant cultivar. Activities of apoplastic peroxidase(s) and H₂O₂ accumulation were observed cytochemically, 3 and 4 h post inoculation, respectively. When digitonin, a O₂·^- elicitor, was infiltrated into cotyledons of resistant and susceptible cultivars, generation of O₂·¯ radicals was shown to be reduced by SOD and inhibited by SHAM and KCN as observed after infection, and also by DPI. Our results strongly suggest that cotton cotyledons contain two O₂·¯- generating systems and that cells undergoing the HR in response to an avirulent race of Xcm produce O₂·¯ through the activation of an apoplastic peroxidase.