In an effort to identify the enzymatic mechanism responsible for the synthesis of reactive oxygen species produced during the hypersensitive response, preparations of rose (Rosa damascena) cell plasma membranes, partially solubilized plasma membrane protein, and cytosol were assayed for the NADH-and NADPH-dependent synthesis of superoxide using assays for the reduction of cytochrome c (Cyt c), assays for the reduction of nitroblue tetrazolium, and assays for the chemiluminescence of N,N-dimethyl-9,9-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue tetrazolium assay to plasma membrane, and the lucigenin assay to the partially solubilized plasma membrane protein (with NADH). This suggests that no two assays measure the same set of enzymes and that none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt c reductase confounded attempts to use Cyt c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the chemiluminescence. Superoxide synthesis detected with lucigenin should be confirmed by superoxide-dismutase-sensitive Cyt c reduction.In plant systems the synthesis of ROS, including superoxide, hydrogen peroxide, and the hydroxyl radical, occurs as a by-product of normal metabolism. However, excesses of ROS are produced during particular periods of development and in response to various stresses. There has been a recent focus on ROS produced during the hypersensitive response to pathogen infection and to the presence of noninfective elicitors from pathogenic and nonpathogenic microbes.One mechanism for the production of ROS is the singleelectron reduction of O 2 to form superoxide. Dismutation of superoxide forms hydrogen peroxide and in the presence of transition metals, principally ferrous iron, the Fenton reaction produces hydroxyl radicals from the substrate hydrogen peroxide. Thus, the formation of superoxide leads to the other species of ROS. The detection of superoxide and the quantification of superoxide synthesis is a challenge, because traditional spectrophotometric methods are not especially sensitive. A sensitive method that depends on the chemiluminescence of lucigenin has been used extensively; it is considered to be a specific indicator of superoxide because it shows little signal from hydrogen peroxide (Corbisier et al., 1987) and has been used in a large number of studies of ROS production (for refs., see Faulkner and Fridovich, 1993).Using lucigenin, our laboratory (Auh and Murphy, 1995) reported the accumulation of superoxide in suspension cultures of rose (Rosa damascena) cells treated with a cell wall elicitor from Phytophthora cinnamomea. Subsequently, Murphy and Auh (1996) used lucigenin to measure the rate of synthesis of superoxide by enzymes in rose cell plasm...

The UVC-induced synthesis of hydrogen peroxide ( HzOz) by cultured rose (Rosa damascena Mill. cv Gloire de Gui-Ian) cells is inhibited by broad-band radiation containing UVA, UVB and blue wavelengths. In order to understand the basis for this inhibition, we tested the effects of broad-band radiation and of monochromatic radiation on NADH-and NADPH-dependent Ozsynthases of purified plasma membranes. Both activities were inhibited by wavelengths from 254 to 435 run, with the NADPHdependent enzyme being more sensitive than the NADHdependent enzyme. Incubation in the presence of flavin adenine dinucleotide had a small but significant ameliorative effect on the inhibition of the Ozsynthase activities of plasma membranes by 300 nm radiation. The data support the hypothesis that the NADH-and NADPH-dependent activities are catalyzed by different enzymes. The NADPH-dependent activity, but not the NADH-dependent activity, was sufficiently sensitive to account for the inhibition of HzOz synthesis by the broad-band radiation.

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Summary. An NADH-specific oxidation reduction enzyme has been partially purified from rose cell microsomes by aqueous twophase partitioning, ultracentrifugation, and ion-exchange chromatography, on the basis of the enzyme's ability to activate lucigenin chemiluminescence in the presence of NADH. The enzyme showed strong similarity to a plasma membrane NADH oxidase ("superoxide synthase" as assayed by lucigenin chemiluminescence; T. M. Murphy and C.-K. Auh, Plant Physiol. 110: 621-629, 1996) in its response to substrate, to Triton X-100, and to diphenylene iodonium, an inhibitor of mammalian neutrophil NADPH oxidase and other fiavoenzymes. However, its fluorescence spectrum was not characteristic of flavins and instead was similar to that of pterins. Thus inhibition of an enzyme-catalyzed reaction by diphenylene iodonium does not necessarily imply that the reaction is catalyzed by NADPH oxidase or another flavoenzyme. Superoxide synthesis catalyzed by the enzyme preparation was very low but could be increased at least twofold by the addition of a quinone, menadione. This suggests the enzyme acting in conjunction with a natural quinone could produce activated oxygen species in stressed plant ceils.