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.

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Coinfiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O 2 ⅐؊ ), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H 2 O 2 infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2.5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O 2 ⅐؊ -generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H 2 O 2 is required for local and systemic accumulation of SA, which may locally control the generation of O 2 ⅐؊ . Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.

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