Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress. -Physiol. Plant. 97: 388-396.The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophically grown, suspension-cultured soybean celts (Glycine max L.). Within two days, 50 to 500 ruW oxyfluorfen, a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants aseorbate and glutathione showed different responses. Under stress conditions with more than 250 nM oxyfluorfen, the eellular ascorbale concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of aseorbate in untreated control cells) increased nearly 3-fold in the presence of 250 TM oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely aseorbate peroxidase, glutathione reduetase, monodehydroascorbate reduetase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 nM, while dehydroascorbate reduetase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.
Suspension‐cultured, photoheterotrophically grown, green soybean cells (Glycine max L.) were used to investigate alterations in the cellular contents of ascorbate and glutathione, as well as specific activities of antioxidative enzymes, elicited by salicylic acid (SA) and BTH [benzo(1,2,3)thiadiazole‐7‐carbothioic acid S‐methylester]. Both antioxidants were positively regulated by 48‐h incubations with SA and BTH, respectively, the latter induced a stronger increase in antioxidant levels compared to SA. The specific activities of glutathione reductase, monodehydroascorbate reductase and glutathione S‐transferase increased strongly in soybean cells as a response to both SA and BTH. The enzyme activations observed were in the range of 2–8‐fold. Catalase activity was also increased 2‐fold by SA but decreased when cells were incubated with BTH. These results indicate an activation of the cellular antioxidative system at both the antioxidant and enzyme level. In addition, the effects of SA and BTH on phytotoxicity exerted by the peroxidizing herbicide oxyfluorfen were investigated. Both compounds protected soybean cells from herbicide‐induced lipid peroxidation in a time‐ and concentration‐dependent manner and strongly suppressed the herbicide‐induced accumulation of protoporphyrin IX. SA as well as BTH antagonize the action of peroxidizing herbicides.
Ketol-acid reductoisomerase (KARI, EC 1.1.1.86) was purified to homogeneity from etiolated barley shoots (Hordeum vulgare) using anion exchange, Red-Sepharose, hydrophobic interaction, and chromatofocusing steps. Purification yielded 0.25 to 0.27 mg of pure KARl per 100 g fresh weight of starting material. l h e specific activity of the purified enzyme was 6 pmol of NADPH oxidized min-' mg-' with acetohydroxybutyrate as substrate. The native enzyme had an apparent molecular weight of 115,000 as estimated by gel filtration and appeared to be a homodimer with a subunit molecular weight of 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. l h e K,,, values of the purified KARl for acetolactate, acetohydroxybutyrate, and NADPH (determined with acetohydroxybutyrate) were 11, 38, and 4.3 p~, respectively. l h e V,,, obtained with acetohydroxybutyrate was 1.8 pmol min-' mg-'; the corresponding value for acetolactate was 0.16 pmol min-' mg-'. l h e enzyme showed optimum activity at pH 7.5. When either acetolactate or acetohydroxybutyrate was used as substrate, the experimental herbicidal compound 2-dimethylphosphinoyl-2-hydroxyacetic acid inhibited the purified KARl in a time-dependent and reversible manner. l h e initial inhibition was strictly competitive. l h e inhibition constant values were 0.46 (using acetolactate as substrate) and 0.19 p~ (acetohydroxybutyrate), respectively.
Both components of nitrogenase, dinitrogenase and dinitrogenase reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of dintrogenase reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of dinitrogenase reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin-and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of dinitrogenase reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of dinitrogenase reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation.
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