Responses of Antioxidants to Paraquat in Pea Leaves (Relationships to Resistance).

Differnential sensitivity to the oxidant paraquat was observed in pea (Pisum sativum L.) based on cultivar and leaf age. To assess contributions of inductive responses of the antioxidant enzymes in short-term resistance to oxidative damage, activities of glutathione reductase (GR), superoxide dismutase (SOD), and ascorbate peroxidase (APX) and transcript levels for plastidic GR, Cu,Zn SOD, and cytosolic APX were determined. Responses to paraquat exposure from three different leaf age classes of pea were studied. Resistance was correlated with leaf age, photosynthetic rates, enzyme activities, and pretreatment levels of plastid GR and plastid Cu,Zn SOD transcripts. In response to paraquat, small increases in activities of GR and APX were observed in the more resistant leaves. These changes were not reflected at the mRNA level for the plastidic GR or Cu,Zn SOD. Paraquat-mediated increases in cytosolic APX mRNA occurred in all leaf types, irrespective of resistance. Developmentally controlled mechanisms determining basal antioxidant enzyme activities, and not inductive responses, appear to be critical factors mediating short-term oxidative stress resistance.

Differential response of Cu,Zn superoxide dismutases in two pea cultivars during a short-term exposure to sulfur dioxide.

Pea cultivars Progress and Nugget have been shown previously to be differentially sensitive with respect to apparent photosynthesis in a short-term exposure to 0.8 microliters/l SO2. One possible contributing factor to the relative insensitivity of apparent photosynthesis of Progress to SO2 is an increase in superoxide dismutase (SOD) activities. We show here that both chloroplastic and cytoplastic Cu,Zn-SOD proteins increased in Progress on exposure to sulfur dioxide whereas both proteins decreased in Nugget. The increase in cytosolic Cu,Zn-SOD protein was greater than that of chloroplastic Cu,Zn-SOD protein. Using a gene-specific probe for the plastid SOD, northern blot analysis revealed an initial decrease in transcript abundance of the chloroplastic Cu,Zn-SOD gene in Progress on exposure to SO2 with an eventual recovery to pre-exposure levels. The transcript levels of the chloroplastic Cu,Zn-SOD decreased in Nugget over the time period of the exposure. These results suggest that a combination of translational and post-translational mechanisms may be involved in SO2-induced changes in cytosolic and plastidic Cu,Zn-SODs in pea.

Purification and characterization of glutathione reductase isozymes specific for the state of cold hardiness of red spruce.

Isozymes of glutathione reductase (GR) have been purified from red spruce (Picea rubens Sarg.) needles. Two isozymes could be separated by anion-exchange chromatography from both nonhardened or cold-hardened tissue. Based on chromatographic elution profiles, the isozymes were designated GR-1NH and GR-2NH in preparations from nonhardened needles, and GR-1H and GR-2H in preparations from hardened needles. N-terminal sequencing and immunological data with antisera obtained against GR-1H and GR-2H established that the isozymes from hardened needles are different gene products and show significant structural differences from each other. Chromatographic, electrophoretic, and immunological data revealed only minor differences between GR-2NH and GR-2H, and it is concluded that these isozymes are very similar or identical. Anion-exchange chromatography and native polyacrylamide gel electrophoresis also established that GR-1NH and GR-1H are different proteins. From these data we conclude that GR-1H is a distinct gene product, present only in hardened needles. Therefore, GR-1H can be considered to be a cold-hardiness-specific GR isozyme, and GR-1NH can be considered to be specific for nonhardened needles. It is proposed that GR-1H is a cold-acclimation protein.

Cold-hardiness-specific glutathione reductase isozymes in red spruce. Thermal dependence of kinetic parameters and possible regulatory mechanisms.

The thermal dependence of kinetic parameters has been determined in purified or partially purified preparations of cold-hardiness-specific glutathione reductase isozymes from red spruce (Picea rubens Sarg.) needles to investigate a possible functional adaptation of these isozymes to environmental temperature. We have previously purified glutathione reductase isozymes specific for nonhardened (GR-1NH) or hardened (GR-1H) needles. Isozymes that were distinct from GR-1NH and GR-1H, but appeared to be very similar to each other, were also purified from nonhardened (GR-2NH) or hardened (GR-2H) needles (A. Hausladen, R.G. Alscher [1994] Plant Physiol 105: 205-213). GR-1NH had 2-fold higher Km values for NADPH and 2- to 4-fold lower Km values for oxidized glutathione (GSSG) than GR-2NH, and a similar difference was found between GR-1H and GR-2H. However, no differences in Km values were found between the hardiness-specific isozymes GR-1NH and GR-1H. There was only a small effect of temperature on the Km(GSSG) of GR-1H and GR-2H, and no significant temperature effect on Km(NADPH) or Km(GSSG) could be found for the other isozymes. These results are discussed with respect to "thermal kinetic windows," and it is proposed that the relative independence of Km values to temperature ensures adequate enzyme function in a species that is exposed to extreme temperature differences in its natural habitat. A variety of substrates has been tested to characterize any further differences among the isozymes, but all isozymes are highly specific for their substrates, NADPH and GSSG. The reversible reductive inactivation by NADPH (redox interconversion) is more pronounced in GR-1H than in GR-2H. Reduced, partially inactive GR-1H is further deactivated by H2O2, whereas GR-2H is fully reactivated by the same treatment. Both isozymes are reactivated by GSSG or reduced glutathione. It is proposed that this property of GR-2H ensures enzyme function under oxidative conditions, and that in vivo the enzyme may exist in its partially inactive form and be activated in the presence of increased levels of GSSG or oxidants.

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves.

Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K(m) for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 mum for plastidic and extraplastidic glutathione reductase, respectively, at 25 degrees C. The K(m) for NADPH was 4.8 and 4.0 mum for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L(-1) ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

Metabolic bases for differences in sensitivity of two pea cultivars to sulfur dioxide.

An oxidative chain reaction of sulfite initiated by the superoxide ion produced in the Mehler reaction has been implicated in the damage of plants exposed to sulfur dioxide. The toxicity of SO(2) may be alleviated by free radical scavenging systems acting to terminate this chain reaction. Hence, the relative sensitivity of plants to SO(2) toxicity could depend on differences in the responses of the levels of antioxidant metabolites and enzymes. The effect of SO(2) exposure on glutathione and ascorbic acid contents, glutathione reductase, and superoxide dismutase activities was assayed in two cultivars (Progress, Nugget) of pea (Pisum sativum L.) in which apparent photosynthesis showed a differential sensitivity to 0.8 microliter per liter SO(2) (R. Alscher, J. L. Bower, W. Zipfel [1987] J Exp Bot 38:99-108). Total and reduced glutathione increased more rapidly and to a greater extent in the insensitive Progress than in the sensitive Nugget, as did glutathione reductase activities. Superoxide dismutase activities increased significantly in Progress, whereas no such change was observed in Nugget as a result of SO(2) exposure. This increase in superoxide dismutase activity was observed at 210 minutes after 0.8 microliter per liter SO(2) concentration had been reached, in marked contrast to the increases in reduced glutathione content and glutathione reductase activity, which were apparent at the 90 minute time point. These data suggest that one basis for the relative insensitivity of the apparent photosynthesis of the pea cultivar Progress to SO(2) is the enhanced response of glutathione reductase, superoxide dismutase activities, and glutathione content.

Response of photosynthesis and cellular antioxidants to ozone in populus leaves.

Atmospheric ozone causes formation of various highly reactive intermediates (e.g. peroxyl and superoxide radicals, H(2)O(2), etc.) in plant tissues. A plant's productivity in environments with ozone may be related to its ability to scavenge the free radicals formed. The effects of ozone on photosynthesis and some free radical scavengers were measured in the fifth emergent leaf of poplars. Clonal poplars (Populus deltoides x Populus cv caudina) were fumigated with 180 parts per billion ozone for 3 hours. Photosynthesis was measured before, during, and after fumigation. During the first 90 minutes of ozone exposure, photosynthetic rates were unaffected but glutathione levels and superoxide dismutase activity increased. After 90 minutes of ozone exposure, photosynthetic rates began to decline while glutathione and superoxide dismutase continued to increase. Total glutathione (reduced plus oxidized) increased in fumigated leaves throughout the exposure period. The ratio of GSH/GSSG also decreased from 12.8 to 1.2 in ozone exposed trees. Superoxide dismutase levels increased twofold in fumigated plants. After 4 hours of ozone exposure, the photosynthetic rate was approximately half that of controls while glutathione levels and superoxide dismutase activity remained above that of the controls. The elevated antioxidant levels were maintained 21 hours after ozone exposure while photosynthetic rates recovered to about 75% of that of controls. Electron transport and NADPH levels remained unaffected by the treatment. Hence, elevated antioxidant metabolism may protect the photosynthetic apparatus during exposure to ozone.

Seasonal changes in antioxidants in red spruce as affected by ozone.

Two-year-old red spruce (Picea rubens Sarg.) seedlings were exposed to various levels of ozone, from 0.4 to 3 times ambient levels, in open-top chambers in Ithaca, NY, USA. Exposures, which varied with changes in day length, were from 30 May to 16 December 1987 and 1 June to 1 December, 1988. During the second exposure period, ascorbic acid, total and oxidized glutathione, α-tocopherol, and superoxide dismutase were measured in current and previous year's needles at monthly intervals from May to July, and at bi-weekly intervals from September to December. Orthogonalized polynomials were used to model the response through time of each variable measured. A one-way analysis of variance model was fitted to every regression coefficient in each polynomial model to test for ozone effects on seasonal patterns of antioxidant levels. Ozone influenced seasonal changes in total glutathione, the oxidized/total glutathione ratio, and α-tocopherol in previous years's needles and α-tocopherol and superoxide dismutase in current year's needles. Averaged over the whole growing season, the oxidized/total glutathione ratio and superoxide dismutase showed an ozone treatment effect in both age classes. Mean total glutathione content increased in previous year's needles, but was not influenced by ozone in current year's needles. Mean oxidized glutathione content was higher with ozone exposure in current year's needles, but not in previous year's needles. The role of antioxidants in cold hardiness and ozone detoxification is discussed.

Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons.

Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 mul/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light.