School of Botany - Theses
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ItemThe control of respiration in carrot-root tissue(University of Melbourne, 1970)1. The 'induced' respiration in carrot-root slices increases in two stages during washing in aerated, distilled water. Stimulation of respiration by DNP and pyruvate indicates that overall respiration rate in freshly cut tissue is regulated by the rate of the pentose phosphate or the glycolytic pathway. 2. Adenine nucleotides and glycolytic intermediates were extracted from phloem parenchyma during development and decline of the induced respiration, and were estimated by enzymatic analysis. The crossover theorem of Chance was used to identify regulatory reactions within pathways of respiration. Changes in respiration rate are correlated with a characteristic sequence of activation of the essentially irreversible reactions of the glycolytic pathway. The first stage of induced respiration corresponds to a period of active synthesis, when ADP produced in the cytoplasm from synthetic processes, stimulates pyruvate kinase. During the second stage, phosphofructokinase is stimulated by ATP. 3. The activity of pyruvate kinase, extracted from freshly cut phloem parenchyma is not affected by physiological concentrations of allosteric effectors of pyruvate kinase extracted from other sources. Hence, stimulation of pyruvate kinase in the first stage of induced respiration may be primarily due to a mass-action effect of increased ADP at the site of the ADP- dependent glycolytic reactions. 4. Exogenously applied adenine and uridine nucleotides, and nucleosides enter the metabolic phase of the carrot cell and markedly stimulate the respiration rate of freshly cut tissue. There is a crossover at pyruvate kinase within several minutes of adding either ADP or ATP, thus confirming the importance of this enzyme during the first phase of induced respiration. 5. The magnitude and direction of the respiration response to cyanide is positively correlated with the initial respiration rate. At rates below a 'critical value of initial respiration', low concentrations of cyanide may stimulate glycolysis by increasing the concentration of cytoplasmic ADP, and thereby stimulate glycolysis. 6. Variation in respiration rates of freshly cut tissue can be explained in terms of a range of endogenous ADP levels in different tissue batches. 7. The results of analyses of intermediates and nucleotides in tissue aging in water, and the effects of nucleotides, nucleosides, DNP, pyruvate and cyanide on respiration rate, glycolytic intermediates and adenine nucleotide levels all support the conclusion that development of induced respiration is controlled by the concentration of cytoplasmic ADP. 8. Although respiration of freshly cut slices is stimulated slightly by KC1 or NaCl, 'salt' respiration proper develops at the end of the second stage of induced respiration in xylem (70 hr) and phloem (30 hr) parenchyma slices. The capacity for salt respiration declines in tissue washed for more than 8 days. 9. A new, steady state of enhanced respiration is recorded within 1. 5 to 3 minutes of adding 50 mM KC1 to well-washed tissue in an O2 electrode system. 10. When the capacity for salt respiration is low, salt induces changes in glycolytic intermediates and adenine nucleotides, but, phosphoglycerokinase is the only enzyme greatly stimulated. 11. In well-washed slices capable of high salt respiration rates, salt alternately stimulates pyruvate kinase, phosphoglycerokinase and phosphofructokinase, and induces large oscillations in the level of total ADP. 12. It is concluded that the effect of salt on the concentration of cytoplasmic ADP at the site of glycolytic kinases is responsible for establishment of salt respiration.