Relative Contribution of Cytochrome - mediated and Cyanide - resistant Electron Transport in Fresh and Aged Potato Slices 1

The respiration of fresh potato ( Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively. The maximal capacity of the cytochrome path (V,yt) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V,yt of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate moblization, with the result that the electron transport capacity of the cytochrome chain is exceeded. The respiration rate of thin potato slices immediately following cutting is four to five times that of the intact tuber. In the subsequent 24 hr the respiration rises another 4to 5-fold to yield the wound-induced respiration. Thus, the respiration ofaged slices may be 25 times that of the intact tuber (18, 32). Slice aging in potato is characterized by two main metabolic transitions. First, whereas tuber and aged slice respiration entails carbohydrate metabolism by way of glycolysis, the pentose-P pathway and the TCAC,3 fresh slices utilize fatty acids as substrate and show little or no TCAC activity (18). Second, slice respiration becomes CNinsensitive with aging (10), and tuber respiration in response to CN progresses from resistant to stimulated (11, 29). By contrast, fresh slice respiration is predominantly CN-sensitive (10). Attempts to correlate the respiratory rise which accompanies slice aging with an increase in specific glycolytic or respiratory enzyme activities have not been successful (16, 33). On the other hand, it has been shown that the control of phospholipid biosynthesis determines the development of the wound-induced respiration (33, 34). These observations implicate membrane biosyn'This work was supported by United States Public Health Service Grant GM 19807 to G. G. L. 2Address reprint requests to Dr. G. G. Laties, Department of Biology, University of California, Los Angeles, Calif. 90024. 3 Abbreviations: CLAM: m-chlorobenzhydroxamic acid; CCCP: carbonyl-cyanide m-chlorophenyl hydrazone; Fp.,: medium potential, absorbing, nonfluorescent flavoprotein; TCAC: tricarboxylic acid cycle. thesis as a crucial element in the development of respiration with slice aging. Efforts to explain the induced respiration by an increase in mitochondria number have also been unsuccessful. The increase in mitochondrial protein accompanying aging in a day is 2-fold or less (7, 10, 31), whereas the respiration increase in the aged slice is 5-fold compared with the fresh slice, and 25-fold compared with the intact tuber. Evidence suggests that the latent Cyt oxidase capacity of fresh slices may be adequate to sustain the wound-induced respiration. Thus, uncouplers elicit a 3to 4-fold increase in fresh slice respiration which, although different in carbon path from the wound-induced respiration, is CN-sensitive (17-19). The absence of TCAC activity in coupled and uncoupled fresh potato slices has been attributed to the inhibition of the cycle rather than to its absence, since mitochondria isolated from fresh slices oxidize TCAC substrates with the same specific oxidative activity demonstrated by mitochondria from aged slices (7, 18). Since the respiration of fresh potato slices is anomalous, aging involves the dissipation of the anomalous respiration as well as the development of the normal induced respiration. Thus, the respiratory increase in interest is much larger than it appears. With the development of a CN-resistant electron transport path with aging (7, 8, 10) the existence of two major pathways in aged slices raises the question of the relative contribution ofeach to the total respiration. When we speak of CN-resistant respiration, is the resistant path operative in the absence of CN, and if so, to what extent? Herein we examine the contribution of the alternate path to the respiration of aged potato slices by a technique heretofore confined to the determination of path distribution in isolated CN-resistant mitochondria (2, 3). The analysis depends on the specific inhibition of the CN-resistant electron transport path by one or another hydroxamic acid-in our case, CLAM (26). MATERUILS AND METHODS Slice Preparation. Potato tubers (Solanum tuberosum var. Russet Burbank) stored at 7 C were halved and pierced within the vascular ring with a 9-mm-diameter stainless steel borer, the cores being left in the tuber pieces. Slices 1 mm thick were cut into ice water from the pierced tuber halves with a microtome blade fixed in an appropriate bed. Slices were rinsed with distilled H20 and either used promptly as fresh slices or set to age in circularly rotated Erlenmeyer flasks in 10 volumes of 0.1 mM CaSO4 at 25 C, the solution being changed at frequent intervals in the first few hr; and five or six times thereafter in a 24or 48-hr period. Bacterial contamination during aging was negligible. Respiratory Measurements. Respiration was measured by conventional Warburg manometry at 25 C. Fourteen slices (about 1 g fr wt) were transferred to standard Warburg flasks containing 4 ml of 0.1 mM CaSO4 with the appropriate respiratory inhibitor at pH 5.5, and containing 0.2 ml of 10% NaOH and a fluted filter paper in the center well. Where CN was used, KCN was added 232 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. CYANIDE-INSENSITIVE RESPIRATION directly to the experimental solution, the pH being adjusted to 5.5 with 0.4 N H2SO4. Six-tenths ml of a suitable mixture of Ca(CN)2Ca(OH)2 (22) was added to the center well. Respiratory rates were obtained over a period of 2 hr. A stock solution of CCCP was made in absolute ethanol. The stock solution was diluted with 10 mm K-phosphate in 0.1 mm CaSO4 to give a final uncoupler concentration of 10 !LM. Ethanol had no effect on the respiration at that dilution. ANALYSIS OF TITRATION DATA Titrations of potato slice respiration with CLAM (26) and CN were carried out as described by Bahr and Bonner (2). In mitochondria the equation describing the total respiration is