Production of antioxidants was investigated in needles of fir (Abies alba Mill.) and spruce (Picea abies (L.) Karst) after exposure to low concentrations of So2, 03, and a combination of both pollutants. Glutathione reacted most sensitively to pollutants followed by vitamin E and vitamin C. In spruce needles, the overall increase of antioxidants after exposure to air pollutants was lower than in needles of fir. SO2 was more potent than 03. Maximum increase of antioxidants was found in needles after exposure of trees to SO2 + 03.Oxidation of both proteins and lipids is among the major phytotoxic consequences of 03 and SO2 action (1, I1, 13). Oxidative degradation of lipids can be easily detected by decomposition products, such as ethane and malonaldehyde (17). Although ethane is a sensitive indicator, a significant increase in gas production is generally associated with visual injury of plants (4). This limits the specific use of ethane as an early indicator of oxidative stress in plants. Antioxidants, such as the vitamins C and E or glutathione, determine the extent of oxidative degradation of cell components (4,17,18 RESULTS AND DISCUSSIONIn spruce, we found a 3-fold higher glutathione level in the filtered air-treated control needles of the youngest shoot than in control needles of fir (Fig. 1). After long-term exposure to different air pollutants, needles neither had visual injury nor elevated lipid oxidation, measured as ethane production. Treatment of fir and spruce with SO2 gave a higher increase of antioxidants in needles than treatment with 03 or charcoal-filtered air. Further, exposure to the pollutants alone significantly increased (P < 0.01) the glutathione content in fir needles compared to filtered air-treated needles. A combination of both gases gave maximum response and significantly enhanced (P < 0.01) the level of vitamin E and glutathione in needles of both fir and spruce compared to the antioxidant levels in needles either to 03 alone or to filtered air. Needles of fir had a 1.9-fold and 3-fold higher content ofvitamin E and glutathione, respectively, after exposure to S02 + 03 than needles exposed to filtered air. In comparison, needles of spruce had only a 1.3-fold higher content of both antioxidants after treatment with S02 + 03 than needles treated with filtered air.The increase of antioxidant levels above the filtered air-treated control was higher in 2-year-old spruce needles, exposed to air pollutants for 2 years, than in the youngest needles, exposed to pollutants for one vegetation period (Fig. 2). In general, the level ofantioxidants above the control was higher after treatment with SO2 or S02 + 03 than after treatment with 03 alone. Increase of glutathione was the most sensitive reaction to air pollutant treatment. No significant increase (P > 0.05) was found for vitamin C after exposure of 1-year-old needles to different pollutants. However, in 2-year-old needles the amount of all antioxidants tested was significantly higher (P < 0.05) after exposure to S02 + 03 than to 03...

Many bleaching herbicides with different core structures inhibit phytoene desaturase (PD), a membrane-bound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40precursor of β-carotene. Prospects are good that new PD-active herbicides will be discovered by screening for bleaching activity. Accordingly, interest in PD enzymology and molecular genetics has increased. Although active carotenogenic cell-free systems are available, no isolation of PD has been achieved since the enzyme cannot be detected in its isolated form due to complete loss of activity. A portion of theRhodobacterPD gene was incorporated into an appropriate plasmid which could be expressed inE. coli.This system was used to produce an antibody specific against PD from higher plants as well asRhodobacter.All PDs assayed had an apparent molecular weight of 52 to 55 kDa. ARhodobactergene probe hybridized with a 3.1 kbBamH I fragment fromAphanocapsawhich allowed us to sequence the PD gene from this cyanobacterium. Its DNA sequence matched with the apparent molecular weight of the PD band in the western blot, and a fusion-gene product was found to be immunoreactive with theRhodobacterPD antibody,Anacystismutants were produced exhibiting cross-resistance against norflurazon and fluorochloridone. Apparently, this resistance is due to an altered PD with concurrent decrease of inhibitor binding affinity. Cloning of the resistant gene into the wild type is in progress.

A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.

The X-ray structure of the T39K mutant of the variable domain of a human immunoglobulin kappa light chain has been determined at room temperature to 1.7 A resolution with a conventional R factor of 0. 182. T39K crystallizes in the triclinic space group P1 [a = 35.4 (1), b = 40.1 (1), c = 43.1 (1) A, alpha = 66.9 (1), beta = 85.4 (1), gamma = 73.8 (1) degrees ]. The unit-cell contains two monomers, related by a non-crystallographic twofold axis. The use of a novel type of local non-crystallographic symmetry restraints on related isotropic displacement parameters and 1-4 distances as incorporated in the refinement program SHELXL improves the model and quality of the maps, but local differences between both monomers in areas subject to different packing contacts can still be observed. 12 overall anisotropic scaling parameters were refined. These may have compensated for the difficulties in accurately scaling single rotation axis image plate data from a triclinic crystal, because of the scarcity of common equivalent reflections. The final model has been used to perform a number of tests on anisotropic scaling, non-crystallographic symmetry, anisotropic refinement, determination of standard uncertainties and bulk solvent correction. It is remarkable that removal of the NCS restraints from the final model caused Rfree to increase. These tests clarify the strategies for optimum use of SHELXL for refinement at medium as opposed to atomic resolution.

To study the role of glutathione reductase in lipid peroxidation, bean leaves (Phaseolus vulgaris) cv Fori were treated with the herbicide acifluorfen-sodium (sodium 5-12-chloro4-(trifluoromethyl)phenoxy-2-nitrobenzoic acid). Acifluorfen is a potent inducer of lipid peroxidation. In beans, decrease of acid-soluble SH-compounds and lipid peroxidation, measured as ethane evolution, were the toxic events after treatment of leaves with acifluorfen. As a primary response to peroxidation, increased production of antioxidants, such as vitamin C and glutathione, was found. This was followed by elevation of glutathione reductase activity. Enhanced activity of the enzyme prevented both further decline of acidsoluble SH-compounds and lipid peroxidation. Increased production of antioxidants and elevated activity of antioxidative enzymes, like glutathione reductase, seem to be a general strategy to limit toxic peroxidation in plants.The deleterious effect of many xenobiotics, including certain p-nitrodiphenyl ether herbicides, is believed to be strong oxidation ofcell components, such as peroxidation ofpolyunsaturated fatty acids in biomembranes via free radical reactions (10, 13). Research to date has shown that peroxidation of lipids is responsible for damage of proteins, DNA, and pigments (11,12,24,26). Protection against phytotoxic peroxidation is achieved by several antioxidants, such as the vitamins E and C or glutathione (3,26). The lipophilic vitamin E, however, seems to be the most effective radical chain-breaking substance (2). It has to be reductively regenerated by water-soluble GSH3 either directly or via a system consisting of GSH and the water-soluble vitamin C (18). To maintain a high level of active GSH, GSSG has to be rapidly reduced. This reaction is catalyzed by the enzyme GR (EC 1.6.4.2) in the presence of NADPH. GR has been isolated from a number of different organisms including higher plants and bacteria (9, 17).Little is known, however, about the physiological role of GR in plants under peroxidative conditions. Halliwell and Foyer (9) proposed that the enzyme is involved in the ascorbate-glutathi- Enzyme Assay. GR activity was assayed spectrophotometrically at 340 nm by oxidation of NADPH as described by Halliwell and Foyer (9). GLO activity was determined by floating disks of primary bean leaves on 10 ml incubation medium in the dark for 18 h. The incubation medium consists of 10 mm sodium phosphate buffer (pH 7.2) containing 20 mm galactonolactone. The activity of the enzyme was then determined by measuring the vitamin C content of the galactonolactone-incubated disks.Analysis. Total glutathione content was determined according to the method of Law et al. (14). The vitamin E and vitamin C content of primary leaves were determined by HPLC technique and dye titration with 2,6-dichlorophenolindophenol, respectively, as described by Finckh and Kunert (3).To determine herbicide-induced ethane evolution, disks of acifluorfen-pretreated primary leaves (0.2 g) were placed into 10 ml vials and seal...