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The effect of frost defoliation upon the respiratory rate of the sugar beet root and the oxidases present in the beet root. Shrimpton, Douglas Malcolm

Abstract

The effect of frost defoliation upon the respiratory rate of the sugar beet root has been determined. The leaves of 140 mature sugar beets were frozen until brittle and the petioles were frozen to within two inches of the crown on August 18, 1957. Twenty-five beet samples were taken one, four, eight, 12 and 16 days after freezing. A cylinder about 3.8 cm. in thickness was cut from the raid-region of each root, i.e. just below the region of greatest diameter. From this cylinder slices 1 mm. in thickness were cut and discs 1 cm. in diameter cut from the slices. Twenty discs were used in each reaction vessel. Nine manometers were used for the tissue from frost defoliated roots and nine for the controls. Respiration was determined as oxygen uptake and measured by Warburg's direct method. One day after defoliation the respiratory rate of defoliated beets was nine uls./hour (13%) higher than that of the controls on a fresh weight basis. From this point the respiratory rate of defoliated beets dropped slowly until after 12 days it was six uls./hour (9%) lower than that of the controls. Between 12 and 16 days after defoliation there was a rapid decrease in respiratory rate to a point 19 uls./hour (28%) lower than the control. On a dry weight basis there was a slight increase in the respiratory rate of defoliated beets until four days after defoliation. After four days there was a rapid decline in respiratory rate until at 16 days the rate was 80 uls./hour (22%) lower than the controls. Throughout the experiment the control beets respired at a fairly uniform rate. A series of selective inhibitors and specific substrates have been applied to tissue from mature sugar beet roots in an attempt to determine the terminal oxidase enzymes present. Potassium cyanide and sodium azide caused almost complete inhibition of oxygen uptake when applied to tissue slices in Warburg manometers. The residual (cyanide stable) respiration was approximately seven uls./hour/gm. fresh weight {10% of total). Sodium diethyldithiocarbamate (0.05M) caused approximately 40% inhibition of oxygen uptake, and resorcinol (0.01M) caused approximately 20% inhibition. Thiourea had no effect on the rate of oxygen uptake. The oxygen uptake by sugar beet root tissue was unaffected by lowering the oxygen partial pressure to 5%. Carbon monoxide (95/5::CO/02) caused approximately 16% inhibition as compared to tissue respiring in a 95/5::N2/02 gas atmosphere. This inhibition was light stable. 8-hydroxyquin-oline had no effect upon the respiration of the sugar beet root. On the basis of this evidence it appears that the terminal oxidase enzymes present are metallo-protein oxidases and that the copper-protein oxidases other than ascorbic acid oxidase are functional in oxygen uptake. Catalase activity was demonstrated by the evolution of 0₂ from H₂0₂. and peroxidase by oxidation of pyrogallol and catechol in the presence of H₂O₂. There was a great increase in oxygen uptake when ascorbic acid was added to tissue slices. This was due, however, to enzymes other than ascorbic acid oxidase. The presence of phenol oxidase was indicated by the increased O₂ consumption when catechol, protocatechuic acid and tyrosine are added to tissue slices. There was also a tendency for cut surfaces of beet roots to darken, particularly in the region of the vascular rings. The addition of hydroquinone and p-phenyl-enediamine to sugar beet tissue also caused an increased oxygen consumption. No oxidation of reduced cytochrome c has been demonstrated. There was no oxidation of glycolic or lactic acids.

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