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 resi stant 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 cytochemica lly, O-2.(-) was detected in cotyledon disks by the cytochrome c reduc tion assay 3 h after inoculation. This activity was inhibited by super oxide dismutase (SOD) and by the peroxidase inhibitors salicylhydroxam ic acid (SHAM) and KCN but not by the NADPH oxidase inhibitor diphenyl eneiodonium chloride (DPI). Strong NADH oxidation activity also was fo und 3 h after inoculation in crude extracts or in apoplastic washing f luid and was dramatically decreased after treatment with SHAM or KCN, NADH oxidation was activated by 2,4-dichlorophenol and MnCl2, indicati ng the involvement of a peroxidase. Activity of cationic peroxidase is oforms (pI 9 to 9.5) constitutively expressed in cotyledons was found to be enhanced 3 h after inoculation in the resistant cultivar, Activi ties of apoplastic peroxidase(s) and H2O2 accumulation were observed c ytochemically, 3 and 4 h post inoculation, respectively. When digitoni n, a O-2.(-) elicitor, was infiltrated into cotyledons of resistant an d susceptible cultivars, generation of O-2.(-) radicals was shown to b e reduced by SOD and inhibited by SHAM and KCN as observed after infec tion, and also by DPI, Our results strongly suggest that cotton cotyle dons contain two O-2.(-)-generating systems and that cells undergoing the HR in response to an avirulent race of Xcm produce O-2.(-) through the activation of an apoplastic peroxidase.