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

Abstract

Excess active oxygen is generated during the hypersensitive reaction (HR), an incompatible reaction of plants to bacterial pathogens. During HR, lipid peroxidation correlates chronologically with production of the oxygen species, superoxide (02-). However MO) seedlings were grown in a growth chamber at 23 to 270C with a photoperiod of 14 h/l0 h light/ dark. The seedlings were grown in vermiculite in plastic trays with cheesecloth covering aeration holes in the bottom. The plants were watered daily from below with 1 mM CaSO4. On days 3 and 5, the CaSO4 was replaced with a nutrient solution consisting of the following: 4 mm CaCl2 2H20, 0.45 mM K2HPO4.3H20, 0.5 mM MgSO4.7H20, 1.25 mM K2SO4, 10 mM NH4NO3, 26 AM Fe citrate, 2.3 AM H3BO3, 0.9 ,AM MnSO4. H20, 0.6 ,uM ZnSO4.7H20, 0.15 ,M CuSO4.5H20, 0.1 M Na2MoO4. 2H20, 0.01 ,uM CoCl2 * 6H20, and 0.1 1 AM NiCl2 * 6H20 (D.G. Blevins, Department of Agronomy, University of Missouri, personal communication). Experiments were performed on 9-to 1 -d-old plants. BacteriaPseudomonas syringae pv. pisi, a bacterium that induces HR on cucumber, was used for all experiments. The bacteria were stored at -70TC in 12% glycerol. When needed, bacteria were removed from the freezer and streaked onto a nutrient agar plate. The bacteria from the plate were transferred to a nutrient agar slant 48 h before inoculation and then were transferred from the slant to nutrient broth 12 to 18 h before inoculation. Bacteria were incubated at 25°C on a reciprocal shaker to obtain a high concentration of cells in the log phase of growth. Preceding inoculation, bacteria were collected by centrifugation at l0,OOOg for 5 min. The bacteria were resuspended in distilled water and collected by centrifugation at l0,OOOg for 5 min. The bacteria were again resuspended in distilled water, and the suspension was adjusted spectrophotometrically to 108 colony forming units mL-'. Plants were inoculated by piercing the abaxial epidermis of the cotyledons 1157 www.plantphysiol.org on April 3, 2019 -Published by Downloaded from

Involvement of Oxidative Processes in the Signaling Mechanisms Leading to the Activation of Glyceollin Synthesis in Soybean (Glycine max)

“…Alternatively, AO species may be able to initiate a lipid peroxidation process in the plant tissues. This has already been described after elicitation (Rogers et al, 1988;Peever and Higgins, 1989;Vera-Estrella et al, 1992) or during incompatible plantlmicroorganism interactions (Keppler and Novacky, 1986;Adam et al, 1989;Popham and Novacky, 1991). The release of signals arising from this early peroxidative event has been postulated (Rogers et al, 1988) and has also been supported recently by studies demonstrating that peroxidized fatty acid metabolites may participate as intracellular messengers in the signaling system that activates various plant defense pathways (Li et al, 1991;Farmer and Ryan, 1992).…”

Responses of two field-grown coffee species to drought and re-hydration

“…Lipoperoxidation was monitored by the spectrophotometric determination of MDA using thiobarbituric acid according to Popham and Novacky (1991). Plant material (1 g FM) was homogenized in 2 cm 3 of trichloroacetic acid, TCA (10 %, m/v) and centrifuged at 15 000×g for 20 min.…”

Measurement of Hydroxyl Radical-Generated Methane Sulfinic Acid by High-Performance Liquid Chromatography and Electrochemical Detection

“…Using the dye-binding technique, various investigators have shown that leaves exposed to paraquat in the light produces hydroxyl radicals in plant tissues (12,14,15,17,18). The results obtained in this study with paraquat treatment are similar to rates of MSA formation reported by others using the dye-binding technique with various plant tissues ( Table 2).…”

“…Only 50-to 100-mg (fresh weight) samples were needed in the paraquat experiment. This is 10-fold smaller than was used with the dye-binding technique (12,14,15). The dye-binding approach requires the elimination of potentially interfering species (sample cleanup) from typical cell and tissue extracts that involve additional steps in the extract preparation.…”

“…This dye-binding assay for MSA has been used successfully for a decade (11)(12)(13)(14), but it would benefit from greater sensitivity and by simplification in the preparation of its extracts from plant and animal tissues. The dye-binding assay requires additional steps during the extract preparation to eliminate interfering substances (11)(12)(13)(14)(15). Because sulfinic acids are fairly strong reducing agents (16), the feasibility of their detection by electrochemical oxidation was investigated here.…”