Activities of the antioxidant enzymes ascorbate peroxidase, catalase, dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, monodehydroascorbate reductase, and superoxide dismutase were assayed in honeydew (Cucumis melo L.) fruit and spinach (Spinacia oleracea L.) leaves either as fresh, frozen to -80 degrees C, frozen in liquid nitrogen, freeze-dried, or acetone powder, representing the various ways tissues are treated prior to enzyme extraction. Treated tissues were analyzed following treatment or stored for up to 8 weeks at -80 degrees C. Enzyme activities in fruit frozen with or without liquid nitrogen and leaves frozen with or without liquid nitrogen or freeze-dried were equal to those of fresh tissue. Enzyme activities in freeze-dried or acetone-powdered fruit and leaves and in acetone-powdered tissues were significantly higher or lower than those in fresh tissue. Enzyme activities in both tissues frozen with or without liquid nitrogen and stored for 8 weeks at -80 degrees C changed little; those in freeze-dried and acetone-powdered tissues, however, significantly increased/decreased over the same period. Fresh tissue should be used in antioxidant enzyme assays, but if storage is necessary, tissues should be placed directly into a -80 degrees C freezer.
Ubiquinone functions primarily in the electron transport chain of the mitochondria of plants and animals. Secondary roles in plant tissue, such as antioxidant activity, have also been proposed. The effect of low temperature exposure on etiolated seedling embryonic axes of two differentially chilling-sensitive species, mung bean (Vigna radiata L.) (chilling-sensitive) and pea (Pisum sativum L. cv. Lincoln) (chilling-tolerant) with respect to respiration rate, lipid peroxidation and ubiquinone content was examined. Whole seedlings (embryonic axis and cotyledon) of both species were exposed to control temperatures (20 C) (6 days) or an acclimatory low temperature treatment of 10 C (3 days) followed by exposure at 5 C (3 days). Measurements were initiated 3 days after seedlings had reached 50% germination (D0). Prior to measurements the cotyledons were removed and only the embryonic axis was used in these experiments.Ubiquinol (UQH 2 ), ubiquinone (UQ) and total ubiquinone (UQ tot ) content decreased in mung bean in response to the temperature treatment and UQH 2 and UQ tot remained stable in the more chilling-tolerant pea. The reduction of the total Q-pool was approximately 85-92%, suggesting a high degree of saturation of the respiration pathways. Respiration declined and the RQ ratio increased in both species in response to low temperature. Cytochrome c oxidase (COX) (EC 1.9.3.1) activity was higher in pea than in mung bean but decreased during low temperature exposure in both species. Considering that levels of MDA (lipid peroxidation) did not increase in either species in response to chilling, decreased levels of UQH 2 and UQ observed in chilling-sensitive mung bean may indicate that these compounds were damaged prior to other membrane lipids during low temperature treatment and rendered undetectable.
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