Success in evaluating effects of a 75% oxygen atmosphere, containing an ambient concentration of carbon dioxide, on levels of protein and oxidative enzymes in cotton, Gossypium herbaceum L. C.B. 1697, leaf tissue was dependent upon generation of genetically uniform and physiologically similar leaves. Sufficient quantities of 2-4 week old leaves for experimental procedures were obtained from plants grown in a 1:1 perlite:vermiculite potting medium supplied with 20% Hoagland's nutrient and maintained in a controlled environment growth chamber under the following conditions: 16 hr day/8 hr night cycle, irradiances of 200-300 µE m⁻² sec⁻¹, 70% humidity, and 30°.

Maximum yields of soluble protein (10-12 mg/g tissue) and active enzymes were obtained when freshly harvested leaves were crushed in liquid nitrogen and then homogenized in 0.1 M Tris-Cl, pH 6.9, containing 0.01 M isoascorbate and polyvinylpyrrolidone [2% (w/v) PVP-10 and 0.5 g dry Polyclar AT/g tissue]. Routinely 90% of the solubilized protein was obtained following centrifugation and chromatography on Sephadex G-50.

Introduction of cotton plants into vinyl chambers of 95% humidity resulted in foliar symptoms of physiological stress, including chlorosis, cellular edema, leaf curling, and loss of turgidity. Of the oxidative enzymes analyzed, catalase activity decreased 30%, glycolate oxidase activity decreased 42%, and peroxidase. increased 89% while soluble protein decreased 27% in plants maintained in the high humidity for 5 days. Exposure of plants to 75% oxygen, 350 ppm carbon dioxide for 48 hr under these conditions resulted in a 70% increase in glutathione reductase and a 25% increase in malate dehydrogenase. Other enzymes, superoxide dismutase, catalase, peroxidase, glyoxylate reductase, and acid phosphatase, appeared to be independent of the oxygen treatment.

When humidity was carefully controlled at 70%, treatment with 75% oxygen, 350 ppm carbon dioxide resulted in a 180% increase in glutathione reductase, an 85% increase in acid phosphatase, and a 22% increase in peroxidase. In the same experiment, catalase decreased by 32% and total soluble protein decreased by 15%. Activities of glyoxylate reductase and both cyanide-sensitive and insensitive superoxide dismutases appeared to be unaffected by the 75% oxygen treatment after 48 hr.

Obviously the impact of humidity as a component of the regulation of metabolism in leaf tissue must be considered. It is concluded that oxidoreductase activities located in the chloroplast, peroxisome, and cytosol are not equally sensitive to high oxygen tensions, and effects of environmental oxygen are not restricted to oxidative enzymes.