Effects of Singlet Oxygen Quenchers and pH on the Bacterially Induced Hypersensitive Reaction in Tobacco Suspension Cell Cultures

Abstract

Suspension cell cultures of Nicotiana tabacum L. inoculated with the incompatible pathogen Pseudomonas syringae pv pisi undergo a hypersensitive reaction. Addition of the singlet oxygen quencher bixin to cell suspensions had no effect on hypersensitive cell death. Addition of the singlet oxygen quencher 1,4-diazabicyclo octane (DABCO) increased the medium pH and delayed the onset of cell death. This delay was eliminated when cell suspensions were buffered, and could also be induced by increasing medium pH with KOH. Bixin and DABCO also did not suppress the hypersensitive reaction in tobacco leaves. These data do not support a role for singlet oxygen in the hypersensitive reaction. Medium pH, however, appears to be a critical factor in cell suspension cultures.Several reports have suggested that active oxygen-induced lipid peroxidation may play a role in the HR,3 a defense reaction commonly induced by plant pathogens following inoculation of incompatible hosts. Keppler and Novacky (10) reported increases in lipid peroxidation during the HR in inoculated cucumber cotyledons and postulated that this increased lipid peroxidation leads to the typical loss of electrolytes during the HR. They further demonstrated that the HRinduced lipid peroxidation could be inhibited by the addition of SOD (1 1 ('02) is an excited state of molecular oxygen which can be generated in a number of ways including the spontaneous dismutation of two 2-radicals, and by the interaction of 02 with the triplet state of photosensitizing compounds such as Chl (7, 13). '02 is a very potent inducer of lipid peroxidation and membrane damage (13). There has been one brief report that 3-carotene (a singlet oxygen quencher) increased bacterial populations in an incompatible host when coinjected into leaves, suggesting an involvement of '02 in the HR (16). The purpose ofthis study was to further investigate a possible role of '02 in the HR.In this study we investigated the effects oftwo singlet oxygen quenchers, DABCO and the carotenoid carboxylic acid bixin, on the HR induced in leaves and in cell suspension cultures of tobacco following inoculation with the incompatible pathogen Pseudomonas syringae pv pisi. Both ofthese compounds are potent 102 quenchers and have been shown to protect tobacco cell suspension cultures against photosensitizer-generated 102 (3). Suspension cultured cells have been used in previous studies of bacterially induced HR (1,2). In this study, changes occurring in cells undergoing the HR were followed by using the fluorescent vital stain FDA (15, 19). Decreased staining of cells by FDA has been correlated with progress of the HR in cell suspension cultures (9). MATERIALS AND METHODS BacteriaCultures ofPseudomonas syringae pv pisi Young, Dye, and Wilkie were originally obtained from R. N. Goodman, University of Missouri. Cultures were grown for 24 h at 28°C on nutrient agar (Difco) with 0.5% yeast extract and 1.0% dextrose (NYDA) at pH 7.0. Bacteria were suspended in sterile water or 50 mM Mes buffer (pH 6.0) and the c...

The generation of oxygen radicals during host plant responses to infection

“…Not surprisingly, a recent study into the effects of singlet oxygen scavengers on the hypersensitive reaction of tobacco cells towards P. syringae pv. pisi found that singlet oxygen was not generated during the host response [107]. ************************** While the hydroxyl radical has been proposed as the radical species most likely to be responsible for initial lesions created in the presence of superoxide-generating systems [55,62], there have been very few studies directed specifically at investigating this possibility in the case of plant-pathogen interactions.…”

“…It is still being suggested [73,107] that singlet oxygen, generated from H0 2 /0 2~ via Equation (6) or by spontaneous dismutation, might play an important role in plant resistance. As discussed earlier in this review, singlet oxygen generation by these routes has been discounted [47,89,97,121].…”

Use of Dimethyl Sulfoxide to Detect Hydroxyl Radical during Bacteria-Induced Hypersensitive Reaction

“…2 Abbreviations: HR, hypersensitive reaction; HO, hydroxyl radical; MSA, methanesulfinic acid; MDA, malondialdehyde. the membrane symptoms of HR (23). Other active oxygen species, such as HO, have been implicated as the cause of cellular damage (5).…”

Chemical Interactions with Bioherbicides to Improve Efficacy

“…This enzyme attacks unsaturated fatty acids in membranes, and produces both singlet oxygen and superoxide (02-). There are indications that singlet oxygen is not directly involved in hypersensitive reactions, but is converted to 02- (209). In some plants, rapid cell death may be caused by disruption of the tonoplast due to lipid peroxidation (64) which releases toxic phenolic compounds previously sequestered.…”