Transgenic tobacco (Nicotiana tabacum cultivar W38) plants that overproduce petunia chloroplastic Cu/Zn superoxide dismutase were exposed to ozone dosages that injure control tobacco plants. Based on foliar injury ratings, there was no consistent protection provided to the transgenic plants. These Ozone damage is thought to result from the action of free radicals and other oxidants produced by the interaction of 03 and its degradation products with plant tissue constituents (9). Cellular antioxidant systems are a front-line defense against oxygen free radicals. The activity of SOD,2 one such enzymatic antioxidant, has been shown to increase in response to 03 (7,8,14). Whether this increase is part of a protective, defense response or is a secondary consequence of injury is subject to debate. Lee and Bennett (12) reported a substantial increase in SOD activity in bean plants treated with EDU, a compound capable of conferring tolerance on 03-susceptible plants (4). They also attributed the "natural" tolerance of young versus old leaves in the 03-susceptible BBL290 bean cultivar to a higher endogenous SOD content in young leaves. They suggested that SOD plays a major role in detoxifying 03-induced superoxide radicals which are at least in part responsible for ozone injury.We (200 ,g/ mL). Three plant types were tested: the control W38. 11, which had been transformed with the kanamycin resistance gene only, and independent transformants SOD 35/1.4 and SOD 13.22 (hereafter called SI and S2, respectively), which overproduced the petunia chloroplastic Cu/Zn-SOD (19). Vegetative plants aged 3 to 4 months with 9 to 11 nonsenescent leaves were used, uniform plants being chosen within each experiment. The newest leaf over 2 cm long was designated as leaf 1. Phenotypically there were no distinguishing features between the control and the two SOD transformants.Ozone fumigation tests were conducted with five plants of each genotype in a dynamic flow system contained within a greenhouse (13); an equal number were placed in a similar chamber minus 03. Ozone was produced by a generator (OREC model 0341-0) supplied with high grade oxygen. Ozone concentration in the chamber was measured by the neutral potassium iodide method (10). A dosage of 0.30 ppm 03 for 6 h was used because it was capable of eliciting a differential response from indicator tobacco cultivars Bel W3 and BelB, sensitive and tolerant, respectively. Forty-eight hours after ozonation, leaves 1 through 8 were rated for visible injury. The experiment was conducted three times. Injury data for leaves 5 through 8 were subjected to analysis of www.plantphysiol.org on May 8, 2018 -Published by Downloaded from

A Cd-binding protein has been isolated from the roots of Cd-treated tomato plants cv. Rutgers. Almost all the Cd from a high-speed supernatant fraction was recovered in a 10,000-dalton fraction from a gel filtration column coincident with 250-nanometer absorbing material. DEAE-cellulose chromatography of this 10,000-dalton material yielded one major component, which eluted at 0.34 molar NaCI, had an absorption spectrum characteristic of metaDlothioein, and showed absorption changes upon acidification typical of metallothionein. Although the Cd-binding protein did not behave like metallothioneins from animal sources during gel electrophoresis at pH 8.9, a single band containing Cd and staining with Coomassie brilliant blue could be detected following electrophoresis at pH 6.9. Synthesis of the Cd-binding protein appeared to be "induced" by treatment of the plants with Cd2". Recent concern over Cd as an environmental contaminant (23) has stimulated research on the response of plants to Cd (7,15,16). Although dose-response data have been obtained for many plant species, little is known concerning the biochemical mechanisms of Cd toxicity or tolerance (19,22,23 Preparation of Cd-Thionein. The high-speed supernatant was fractionated via gel filtration using Sephadex G-50 fine (Pharmacia Fine Chemicals). The column was equilibrated and the sample eluted with 50 mM Tris (pH 7.8). The appropriate fractions were combined and then subjected to DEAE-cellulose chromatography (Whatman DE52). The DEAE-cellulose column was equilibrated with 50 mM Tris (pH 7.8) and, after sample application, washed with the same buffer. The sample was eluted with 200 ml of a linear gradient of 0.20 to 0.40 M NaCl in 50 mm Tris (pH 7.8). The appropriate fractions were combined and then desalted with coarse Sephadex G-25 using deionized H20 as eluant. The sample then was concentrated by ultrafiltration using an Amicon UM-2 filter. Column dimensions are indicated in the figure legends.Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis was via tube gels, 10 cm long and 0.5 cm in diameter. The running gel was 12% acrylamide (0.4% bisacrylamide) and contained 0.03% (v/v) TEMED5, 0.0 175% (w/v) ammonium persulfate, and 0.38 M bis-Tris adjusted to pH 6.9 with HC1. The stacking gel was 2.5% acrylamide (0.625% bisacrylamide) and contained 23% (w/v) sucrose, 0.0625% (v/v) TEMED, 0.07% (w/v) ammonium persulfate, and 62.5 mM bis-Tris adjusted to pH 5.9 with HC1. The reservoir buffer was 20 mm bis-Tris adjusted to pH 6.5 with glutamic acid. Electrophoresis was at room temperature with DNP-aspartate as "tracking dye." Prior to electrophoresis, samples were mixed with an equal volume of sample buffer consisting of 32% (w/v) sucrose and 100 mm bis-Tris adjusted to pH 5.9 with HC1. Duplicate gels were run. One was stained at 5 Abbreviations: bis-Tris: bis(2-Hydroxyethyl)imino-tris(Hydroxymethyl) methane; DNP-aspartate: 2,4-dinitrophenyl aspartate;

It has been proposed that the mode of action of ethylenediurea, a very effective antiozonant, is via an increase in the antioxidant enzyme superoxide dismutase (EH Lee, IH Bennett [1982] Plant Physiol 69: 1444-1449). Data presented here refute that hypothesis. No ethylenediurea-associated increases in Cu/Zn-superoxide dismutase or Mn-superoxide dismutase activity, nor in steady-state Cu/Zn-superoxide dismutase protein levels, were found in soluble extracts of bean (Phaseolus vulgaris L. cv Bush Blue Lake 290) leaves. However, the cytosolic Cu/Zn-superoxide dismutase increased as a result of ozone fumigation and subsequent injury. Also noted was a developmentally related difference between chloroplastic and cytosolic Cu/Zn-superoxide dismutase, the latter declining during maturation of the leaf. SOD in leaves is a primary scavenger for superoxide radicals generated both as a by-product of normal physiological activities and from exposure to pollutants such as ozone. Its importance as an antioxidant is demonstrated by the fact that it has been found in all aerobic organisms studied to date; also, mutants that are deficient in SOD are extremely sensitive to oxidative stress (11). Further, SOD activity increases in response to various oxidative stress conditions (2, 11, 23), including ozone (8,20). Cu/Zn-SOD has been the focus of much of our research; therefore, based on Lee and Bennett's (17) report, we attempted to use EDU to study the regulation of SOD gene expression. However, we found no effect of EDU on SOD activity in our model system, pea, even though the plants were completely protected from ozone damage at doses comparable to those used in bean (Y. Shaaltiel, L.H. Pitcher, unpublished data). Thinking the effect might be genotype related, we studied the species and variety used by Lee and Bennett. Our goal was to test the hypothesis that the protective action of EDU was singularly dependent upon an increase in SOD. Our specific objectives were as follows: (a) to determine if EDU and/or ozone caused a significant change in SOD activity associated with cyt or chl Cu/Zn isoforms, or in Mn-SOD activity during a time period associated with EDU-induced ozone tolerance; (b) to determine if EDU and/ or ozone caused a significant change in the steady-state levels of Cu/Zn-SOD immunologically detectable protein associated with the cyt or chl isoforms during a time period associated with EDU-induced ozone tolerance; and (c) to determine the long-term effects of EDU on overall SOD activity, as well as upon the relative activities of the various isozymes, by following SOD activity in leaves at different stages of development when EDU was administered to the plants.Of major importance to the achievement of our objectives was the employment of two independent methods for the measurement of SOD, each one superior to colorimetric solution assays of leaf extracts or partially purified enzyme as 1388 www.plantphysiol.org on March 24, 2019 -Published by Downloaded from

No abstract

The effect of 0.25 to 0.30 microliter per liter ozone on photosynthesis and respiration and on the ATP and total adenylate content of the primary leaves of pinto beans (Phaseolus vulgaris L.) was examined. Changes in these parameters over a 72-hour time period were correlated with the development of symptoms of ozone toxicity. Toxicity symptoms normally appeared within 24 hours. The content of ATP and total adenylates increased immediately following a 3-hour exposure to ozone. Photosynthesis was depressed initially, but returned to normal within 24 hours. Respiration was not always altered initially, but it was significantly stimulated within 24 hours. We interpret the results to mean that the changes in adenylate content and photosynthesis are early events in the initiation of ozone damage and that the change in respiration is a consequence rather than a cause of cellular injury. Both the P/O ratio as a measurement of oxidative phosphorylation and the spectrophotometric method for ATP analysis which were used in the above studies are subject to quantitative and qualitative errors. Using the more sensitive and specific luciferin-luciferase reaction, we tested the hypothesis that ozone affects ATP levels in bean tissue. By concurrently measuring respiration and photosynthesis rates, we hoped to determine whether changes in ATP and/ or total adenylate concentrations were related to either of these processes. MATERIALS AND METHODSUniform seedlings of Phaseolus vulgaris L. var. Pinto were grown in soil, two per 4-inch clay pot, in an air-filtered greenhouse during the months of March through May at temperatures ranging from 16 C at night to 27 C during the day. Approximately 2 weeks after germination, when the primary leaves were expanded to three-fourths of their final size, the plants were placed in an 8.2 m3 chamber and exposed to 0.25 to 0.30 ,ul/l ozone for 3 hr by a method previously described by Leone et al. (4). Control plants were placed in a similar chamber with a charcoal-filtered air supply. Each experiment included four replicate control and experimental plants.Following ozonation the plants were removed from the chamber and three of the four primary leaves per pot were harvested. Two leaves were harvested immediately for respiration and photosynthesis measurements. The third leaf was frozen in liquid nitrogen and stored at -20 C for analysis of ATP and total adenylate. The primary leaf remaining on the plant served as an indicator of the degree of visible injury induced by the ozone exposure.Respiration and photosynthesis were measured manometrically with a Gilson differential respirometer using Klinker's leaf disc method (2). The results were reported as 1pd oxygen absorbed per hr per mg dry weight of tissue. Respiration rates reflect oxygen uptake in the dark and photosynthesis rates reflect oxygen evolution of tissue exposed to 800 ft-c of light. Photorespiration could not be measured by this manometric procedure.ATP was analyzed by the luciferin-luciferase technique of Strehler (9, 10). A B...