Effects of Carbon Dioxide on Activity of Apple Mitochondria

Plant Physiol. (1973) 51, 1095-1098

Received for publication September 12, 1972

MICHAEL R. SHIPWAY' AND WILLIAM J. BRAMLAGE Department of Plant and Soil Sciences, University of Massachusetts, Amherst, Massachusetts 01002

ABSTRACT been suggested (8) that excess CO2 inhibits mitochondrial oxidations, thereby diverting pyruvate to acetaldehyde and ethanol Effects of CO2 on mitochondrial activity of apple (Malus formation, substances known to accumulate in apples under pumila Mill. var. Richared Delicious) were studied in two excess CO- even though ample 02 is present (26). These sub- ways. Immediate effects were determined by imposing 3 to stances can be toxic in apples (24) and could be the proximal 18% CO2-bicarbonate mixtures on isolated apple mitochon- cause of CO2 injury to them. dria, and long term effects were determined by extracting There is no direct evidence that CO, influences metabolism mitochondria from apples that had been stored for intervals in in apple mitochondria, but some evidence to this effect has atmospheres containing 6 or 12% CO2 plus 3% 02. The CO2- been found in other plant systems. In Ricinus mitochondria, bicarbonate systems had immediate and broad effects on mito- CO2-bi- chondrial oxidations: 18 % C02 stimulated malate oxidation oxidation of succinate to fumarate was retarded by and NADH carbonate mixtures containing more than 10% CO2 (2, 4). about 10%; suppressed a-ketoglutarate, citrate, There appeared to be competitive inhibition by succinic de- oxidations about 10%; and suppressed fumarate, pyruvate, in Iris and succinate oxidations about 32%. The effects of lower CO2 hydrogenase by the CO2 (3). The succinoxidase system concentrations varied with substrates. Mitochondria isolated rhizomes was sensitive to as low as 6% CO2 (6). In addition, from fruit stored in 6 or 12 % CO2 possessed a reduced capacity higher concentrations of CO2, especially above 40%, inhibited to oxidize added succinate or NADH, but retained a marked the NAD-cytochrome c-reductase system and cytochrome sensitivity to C02-bicarbonate mixtures. Respiratory control c-oxidase (3). The sensitivity of cytochrome c-oxidase to CO2 in these mitochondria was somewhat reduced, but CO2 had not has been shown in several other plant species also (15). acted as a strong uncoupling agent. Miller and Hsu (16) showed more far reaching effects of C02-bicarbonate mixtures than had previously been reported. They found concentrations of 15% CO2 to inhibit oxidations of citrate, isocitrate, malate, succinate, and NADH by cauliflower mitochondria. Phosphorylation was also inhibited by 15% CO2. This paper reports an investigation of the effects of increasing concentrations of CO2 on the metabolism of apple mito- directly Saussure (23) demonstrated in 1804 that plants may be in- chondria. Effects of C02-bicarbonate systems imposed the on mitochondria and of CO2-02 atmospheres imposed on whole jured by exposure to high concentrations of CO2. Although been determined. precise nature of CO2 injury has never been characterized, fruit for extended periods have many responses of whole plants and excised plant parts have since been reported. One response often found is a suppression MATERIALS AND METHODS of respiration by high CO2. This was first recorded by Kidd (12) and served as a basis for the development of controlled- Preclimacteric apple fruits (Malus pumila Mill. var. atmosphere storage of apples and pears, since a moderate sup- Richared Delicious) stored for short periods at 0 C in air were pression of respiration by CO2 is associated with a marked used for studies involving the effects of C02-bicarbonate retardation of senescence of these fruits. However, excessive mixtures on mitochondrial activity. Other samples of these CO2 causes injury to fruit, the effective concentration varying fruit were placed in controlled atmospheres containing either with kind of fruit, variety, temperature, and time of exposure. 6% CO2 and 3% 02, 12% CO2 and 3% 02, or air as a control. In apples, CO2 injury initially appears as a brown, dry area in These fruit were stored for up to 16 weeks at 0 C and 90% and around the core, and progressive deterioration of the fruit relative humidity. follows. Apples were peeled and 350 to 400 g of tissue were gently Despite numerous reports of a suppression of respiration in grated into 750 ml of extraction medium using apparatus simi- plants by CO2, little is known of its role in this process. Hulme lar to that described by Romani et al. (21), except that a plastic (9) reported that apples injured by CO2 accumulated succinic grating surface was substituted for a stainless steel screen. acid. In pears, high CO2 caused succinic and citric acids to Extraction was at 0 to 4 C in prechilled apparatus. The me- accumulate (28); CO2 also appeared to slow malate depletion, dium, maintained at pH 7.8. consisted of 0.4 M sucrose, 20 mM an effect later demonstrated in apples (13). Many other reports citrate, 10 mM KH2PO4, 0.75% (w/v) polyvinyl-pyrrolidone indicate changes in organic acid metabolism in various plant (PVP-40), 10 mM EDTA, and 10 mm cysteine hydrochloride species due to excess CO2. (added immediately prior to extraction). The extract was These findings suggest that CO2 can act as a controlling fac- strained through 50-p mesh nylon fabric to remove the major tor in the operation of the tricarboxylic acid cycle, and it has portion of starch present (17, 22), and the filtrate was centrifuged for 10 min at 37,000g. The pellet was resuspended in 30 ml of medium containing 0.4 M sucrose, 10 mM EDTA, and 1 Present address: Kirton Experimental Horticultural Station, 0.1 M tris-HCl at pH 7.5. The homogenate was centrifuged for Boston, Lincolnshire, England. 10 min at 1000g, and the supernatant was again centrifuged at 1095

Downloaded from on January 13, 2020 - Published by www.plantphysiol.org Copyright © 1973 American Society of Plant Biologists. All rights reserved. 1096 SHIPWAY AND BRAMLAGE Plant Physiol. Vol. 51, 1973 Table I. Oxidationt Rates anid Respiratory Conttrol Ratios of to assay. Reaction times did not exceed 5 min. Respiratory Mitochonidria Isolated from Apple Fruit control values were determined following addition of 0.3 Reaction mixtures contained: 0.25 M sucrose; 5 mM MgCI2; 10 ,tmoles of ADP. mM K2HPO4-KH2PO4; 10 mNi tris-HCl, pH 7.2; 3 mg/ml bovine To determine the effects of CO2 on mitochondria, aqueous serum albumin; 2 mg/ml yeast extract: and substrate as indicated. solutions of NaHCO. were prepared in molar concentrations Respiratory control ratio was determined after addition of 0.3 calculated by the Henderson-Hasselbach equation to produce ,umole of ADP. pH 7.20 at 28 C when in equilibrium with 3, 6, 12, and 18% CO2 (27). CO2 gas was slowly bubbled through the solutions for Substrate Substrate Concn ~Rate SUptakeof State 3 02 ControlRespiratoryRatio approximately 10 min until the pH was maintained at 7.20. These solutions were then used to prepare the oxidation media. zttosnIsIin ?ng Changes in pH due to loss of CO2 from the medium during I )ga pretese state 3/state 4 preparation and assay were minimal (less than 0.05 pH unit at Citrate 16 25 ± 3 1.7 ± 0.0 18% CO.). Fumarate 16 35 ± 1 2.4 + 0.3 Pyruvate 16 40 4 6 2.2 ± 0.1 RESULTS -Ketocylutarate 16 48 4--3 2 R 0 3 Mallate 32 59 + 4 1.7 i 0.3 Mitochondrial Activity. The mitochondria obtained from the Succinate 216 77 i 0 2.8 i 0.2 apple fruit were capable of oxidizing NADH and all the tri- NA,DH 1 162 ± 15 2.0 ± 0.0 carboxylic acid cycle acids tested (Table I). Activity remained constant for at least 3 hr after extraction, during which time all assays were completed. Mitochondrial integrity appeared to be similar to that demonstrated for apples by Wiskich (29), except A z i MALATE| that our respiratory control ratios were higher (Table I). ATPase activity (25) of the mitochondria was relatively low (about 0.1 ,umole Pi released/min-mg protein). Effects of CO2-Bicarbonate Mixtures on Mitochondrial Ac- LU y tivity. Oxidation rates in various CO2-bicarbonate mixtures, - INADH adjusted for any "salt effect" (6) (which was about 10% inhibi- CITRAstion-i - at 18% CO2 and 0% at 3% CO2) are shown in Figure 1. z a-KETO These rates were established within a few seconds after addi- w (0 RATE tion of the mitochondria and were linear until ADP became x limiting. Preliminary experiments with succinate indicated that 0 B Y, CO2 had no effect on the respiratory control ratio. z 0 Increasing concentrations of CO2 influenced oxidations of all substrates tested. With malate, a small stimulation was ef- w fected (Fig. 1A). This stimulation appeared to be saturated at LU. low CO2 concentrations, there being no significant difference w among the effects of 6, 12, or 18% CO2. In contrast, the oxida- Ol FUMARATE tions of succinate, fumarate, and pyruvate were markedly sup- PYRUVATE pressed by increasing levels of CO2 (Fig. 1B). An almost linear response between 3% CO2 and 18% CO2 was observed, in- l l I SUCCINA creasing to about a 30% inhibition at the 18% CO2 level for 0 3 6 9 2 15 I8 all substrates. This inhibition was statistically significant at 6% CO2 or greater for all three substrates and was significant % CO2 -BICARBONATE at 3% CO2 for succinate and fumarate.

FIG. 1. Effects of CO2-bicarbonate mixtures on state 3 oxida- Changes in Succinate and NADH Oxidation in Stored Fruit. tion ratLes of apple mitochondria. Reaction media as in Table I To assess the long term effects of CO2 in the atmosphere sur- except Ithat media were made up in indicated % C02-bicarbonate rounding intact fruit, mitochondrial oxidations of succinate mixtures. A: Substrates malate, 32 mM; NADH, 1 mM; citrate, 16 and NADH, the most actively oxidized substrates (Table I) mM; aind a-ketoglutarate, 16 mM; B: substrates succinate, fu- were determined after different storage intervals. Marked marate, and pyruvate, all 16 mM. changes occurred with time in air-stored apples (Tables II and III). Succinate oxidation rose during the first 4 weeks of stor- 37,000, g for 10 min. The pellet was suspended in 2 ml of 0.2 age, leveled off for 2 weeks, and then declined steadily during M sucrn Dse at pH 7.2. This gave a 3-ml mitochondrial prepara- the rest of the storage period. NADH oxidation was not de- tion generally containing 30 to 70 mg of protein, determined termined during the time of the rise in activity, but it exhibited by the Lowry method (14). Total extraction time was 1.25 to a decline with extended time in storage. The rise and subse- 1.5 hr. quent decline in mitochondrial respiration closely resembled Oxidlation rates and respiratory control ratios were measured the respiratory climacteric, to which it probably corresponded polarog;raphically at 28 C with a recording oxygen cathode (11). Mitochondria from fruit stored in 12% CO2 and 3% 02 (Oxygraph Model KM, Gilson Medical Electronics) fitted with also exhibited the rise and fall in oxidation of succinate, but the a Clarn (e-type electrode (Yellow Springs Instrument Co.) and rise was suppressed and the decline was accelerated (Table II); Teflon (0.001 inch) membrane in a 1.5-ml cuvette. The reaction furthermore, the decline in NADH oxidation was accelerated mediunn, following Bonner (5). consisted of 0.25 M sucrose, 5 by this storage atmosphere (Table III). Storage in 6% CO2 mM MIgCl2, 10 mm KH2PO,-K2HPO, buffer, 10 mM tris-HCl and 3% 02 appeared to have little or no effect on succinate buffer ((pH 7.2), and substrate at 16 mm (except NADH, 1 mM oxidation, but to increase the decline in NADH oxidation. and madlate, 32 mM). Also, bovine serum albumin (3 mg/ml) To see whether the effect of CO2 in the atmosphere had a and yeoast extract (2 mg/ml) (10) were added immediately prior persistent effect on mitochondrial activity, after 16 weeks in

Downloaded from on January 13, 2020 - Published by www.plantphysiol.org Copyright © 1973 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 51, 1973 CO2 ON MITOCHONDRIAL ACTIVITY 1097 the designated atmospheres all fruit were stored for an addi- Table IV. Respiratory Conitrol Ratios dutring Oxidationi of tional 4 weeks in air. This transfer did not change the trends Different Substrates b'y Mitochondria from of activity that were established during the preceding 16 weeks. Stored Apples If anything, the transfer to air intensified the decline in mito- Experimental conditions as for Table II, except that 0.3 Amole chondrial activity associated with the preceding exposures to ADP was added at intervals throughout the reaction. C02- Changes in Respiratory Control Ratios in Stored Fruit. Since Substrate CO2 has been reported to affect oxidative phosphorylation (2), Storage in respiratory control of mitochondria from stored apples was Air at 0 C Succinate, NADH, ca-Ketogluta- Fumarate, assessed. The respiratory control ratio is the ADP-stimulated 16 mm 1 mm rate, 16 mm 16 mm state 3 oxidation rate divided by the state 4 oxidation rate (7). weeks state 3 Istate 4 By using four different substrates, in each case respiratory con- 0 2.8 ± 0.2 2.0 4 0.012.8 ± 0.3 2.4 ± 0.3 trol declined during storage in air at 0 C (Table IV). Yet, the 8 2.3 ± 0.1 1.8 0.0 mitochondria clearly retained relatively strong respiratory con- 16 1.9 4 0.1 1.5 ± 0.0 1.3 i 0.0 1.4 + 0.1 trol even at the end of the experiment. 18 2.0 4 0.0 1.6 ± 0.0 1.6 4 0.2 1.7 4 0.3 Respiratory control was affected by storage in 12% CO2 and 3% 02 (Table V). During the 6- to 12-week period of storage, the mitochondria from the 12% CO2 fruit were less tightly Table V. Effects of C02 durinig Apple Storage on Respiratory coupled than those from air-stored fruit. However, after 16 Control of Extracted Mitochondria weeks there was no longer any significant difference between Experimental conditions as for Table IV, except that 16 mm suc- fruit from air or 12% CO2 and 3% 02, and the respiratory cinate was the only substrate used. control values were still relatively high for apple mitochondria. Thus, it appears that CO2 imposed an earlier decline in respira- Storage Atmosphere tory control but did not markedly disrupt oxidative phos- Storage at 0 C phorylation. Air 12% C02, 3% 02 Having established that mitochondria from freshly harvested apples are sensitive to CO2 (Fig. 1), it was of interest to de- weeks state 3/state 4 termine the effects of storage time and atmosphere on this 0 2.8 ± 0.2 2.8 ± 0.2 4 2.4 ± 0.0 2.5 ± 0.3 6 2.6 ± 0.0 2.1 ± 0.0 Table II. Rates of 02 Uptake durinig Succinate Oxidationz by 8 2.3 ± 0.1 2.0 ± 0.1 Extracted Mitochondria from Stored Apples 12 2.7 0.1 2.2 0.1 Mitochondria were extracted after indicated storage time of 14 1.9 ± 0.1 fruit in different atmospheres. Fruits were stored in indicated at- 16 1.9 0.1 2.0 ±4 0.1 mosphere for up to 16 weeks, and some were kept for an additional 4 weeks in air (20-week samples). Reaction mixtures as in Table 1. All values are for state 3 rate of oxidation. Table VI. Effects of 12%-G anid 18%- CO2 Bicarbonate Systems on Succinate Oxidation by Mitochotidria Extracted from Apples Storage Atmosphere Storedfor Initervals in Differenzt Atmospheres Storage at O C Experimental conditions as for Table II, except that reaction Air 6% C02, 12% C02, 3%7o 02 3% 02 medium made up in a 12% or 18%- CO2 bicarbonate mixture. weeks ng atoms/min-mg protein Storage Atmosphere 0 77 ±+0o 77 0o 77 0 4 142 ±t 5 - 110 13 Storage at 0 C Air 12% C02, 3c%0 02 6 146 8 - 101 ± 10 8 106 30 - 98 2 12% 18%c, 12% 18% 12 113 ± 8 145 ±t 15 84 15 16 74 3 72 ± 9 58 4 weeks c e ffect on oxidation rate 20 50 1 39 ± 8 24 1 0 -19 (±1) -31 (±2) -19 (-1) -31 (+2) 4 -14 (+1) -22 (±1) -20 (±3) -35 (I1) 6 -13 (+2) -19 (±1) -20 (+3) -26 (+1) Table III. Rates of 02 Uptake dutring NADH oxidation 8 -7 (±1) -18 (±1) -13 (±3) -26 (±2) by Mitochon0dria Extracted from Stored Apples 12 -7 (i3) -19 (±0) -14 (i2) -23 (±2) I -15 I Experimental conditions as for Table II. 14 -7 (±1) (±1) 16 -11 (i2) -12 (I1) -12 (+4) -16 (±2) Storage Atmosphere Storage at 0 C Air 6% 02, 12% C02, 3% 02 sensitivity. Since both activity and CO2 sensitivity were high 3% 02 with succinate as substrate, mitochondria extracted at different weeks ng atoms/min-mg protein intervals were assayed on succinate in 12% and 18% CO2- 0 162 ± 15 162 ±t 15 162 ± 15 bicarbonate systems. Mitochondria from air-stored fruit lost at 8 166 ± 36 132 ± 9 least 50% of their sensitivity to CO2 by the end of the experi- 12 169 + 17 159 ± 6 116 ± 9 ment (Table VI). Mitochondria from fruit stored in 12% CO2 16 125 ± 5 84 ± 10 89 ± 3 and 3% 02 also lost at least 50% of their sensitivity, but the 20 102 ± 2 60 ± 20 37 ± 1 loss occurred at a slower rate. During the first 12 weeks of storage, mitochondria from the 12% CO2-stored fruit were al-

Downloaded from on January 13, 2020 - Published by www.plantphysiol.org Copyright © 1973 American Society of Plant Biologists. All rights reserved. 1098 SHIPWAY AND BRAMLAGE Plant Physiol. Vol. 51, 1973 ways more sensitive to CO2 than those from air-stored apples. LITERATURE CITED It is evident that 12% CO, in the atmosphere surrounding in- 1. ATKINSON, D. E. 1965. Biological feedback control at the molecular level. tact apples did not reduce the sensitivity of extracted mito- Science 150: 851-858. chondria to CO2 inhibition, but that this sensitivity declined 2. BENDALL, D. S., S. L. RANSON, AND D. A. WALKER. 1958. Some effects of carbon dioxide-bicarbonate mixtures on the oxidation and reduction of cyto- with fruit senescence. chrome c by Ricinus mitochondria. Nature 181: 133-134. Occurrence of CO2 Injury in Fruit. After 12 and 16 weeks in 3. BENDALL, D. S., S. L. RANSON, AND D. A. WALKER. 1960. Effects of C02 on the storage, about 10% of the apples held in 12% CO2 and 3% oxidation of succinate and reduced diphosphopyridine nucleotide by Ricinus 02 exhibited CO2 injury in the core area, but all fruit from 6% mitochondria. Biochem. J. 76: 220-225. 4. BONNER, W. D.. JR. 1950. The succinic oxidase system and its relation to CO2 and 3% 02 or air were free of injury. After 16 weeks in phosphate and bicarbonate. Nature 165: 757-758. the test atmospheres plus 4 weeks in air, 90% of the fruit from 5. BON-NER, W. D., JR. 1967. A general method for the preparation of plant mi- 12% CO2 and 3% 02 and 85% of those from 6% CO2 and 3% tochondria. In: R. E. Estabrook and M. E. Pullman, eds., Methods in En- 02 possessed injury symptoms, whereas all of those stored in zymology, Vol. X. Academic Press, New York. pp. 126-133. 6. BROWN, A., D. BOULTER, AND D. A. COULT. 1968. The influence of C02 on the air remained free of core browning. metabolism of rhizome tissue in Iris pseudocarpus. Physiol. Plant. 21: 271- 281. DISCUSSION 7. CHANCE, B. AND G. R. WILLIANIS. 1956. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17: 65-134. These experiments demonstrate that CO2 can have a strong 8. FAUST, AI., C. B. SHEAR, AND M. W. WILLIASIS. 1969. Disorders of carbolhy- effect on apple mitochondrial activity. CO2-bi- drate metabolism of apples. Bot. Rev. 35: 169-194. controlling 9. HULMIE, A. C. 1956. Carbon dioxide injury and the presence of succinic acid in carbonate systems modified without any lag period the oxida- apples. Nature 178: 218-219. tion of all seven substrates tested. The suppressed oxidations of 10. HtTLME, A. C., J. D. JONES, AND L. S. C. WOOLTORTON. 1964. Mitochondrial succinate and citrate could explain the reported accumulations preparation from the fruit of apple. I. Preparation and general activity. Phytochemistry 3:173-188. of these acids under high CO2 (9, 28). However, the mild stimu- 11. JONES, J. D., A. C. HULME, AND L. S. C. WOOLTORTON. 1965. The respiratory lation of malate oxidation suggests that the effect of high CO2 climacteric in apple fruits. Biochemical changes occurring during the devel- in delaying malate consumption in apples (13) is an indirect opment of the climacteric in fruit detached from the tree. New Phytol. 64: response, probably due to an over-all reduction in mito- 158-167. 12. KIDD, F. 1915. The controlling influence of CO-III. The retarding effect of chondrial activity. carbon dioxide on respiration. Proc. Roy. Soc. Series B Biol. Sci. 89:136-156. The effect of CO2 on mitochondria clearly was not due to an 13. KOLLAS, D. A. 1964. Preliminary investigation of the influence of controlled effect on a single enzyme, such as succinic dehydrogenase, al- atmosphere storage on the organic acids of apples. Nature 204: 758-759. 14. LOWRY, D. H., N. J. ROSEBROUGH, A. L. FARR, AND R.J. RANDALL. 1951. Pro- though the succinoxidase system in apple mitochondria is very teinimeasurement with the Folin Phenol reagent. J. Biol. Chem. 193: 265- sensitive to CO2. Many enzyme systems must be sensitive to 273. CO2 and to different degrees. Such a widespread effect of CO2 15. MILLER, G. W. AND H. J. EVAN-S. 1956. Inhibition of plant cytochrome oxidase on mitochondrial metabolism could be attributed to either by bicarbonate. N'ature 178: 974-976. 16. MIILLER, G. W. AND W. H. Hsu. 1965. Effects of carbon dioxide-bicarbonate or conformational changes of the mitochondrion, or structural mixtures on oxidative phosphorylation by cauliflower mitochondria. Bio- to pH changes within it. Although the pH of the assay medium chem. J. 97: 615-619. was maintained at a constant level for all CO2 concentrations, 17. PALMER,J.M. 1967. Rapid isolation of active mitochondria from plant tissue. CO2 diffuses freely through membranes (18) and could change Nature 216: 1208. 18. RABINOWITCH, E. I. 1945. Photosynthesis and Related Processes, Vol. 1. Inter- the internal pH, thus affecting enzyme activities and oxidation science Publishers, Inc., New York. pp. 172-212. rates. This change should occur very quickly, and the responses 19. RANSON', S. L., D. A. WALKER, ANDI. D. CLARKE. 1957. Effects of C02 on mi- shown in Figure 1 occurred without any lag period. tochondrial enzymes from Ricinus. Biochem. J. 66: 57. Long term exposure of intact fruit to CO2 evoked changes in 20. RANSON, S. L., D. A.WNALKER, AND I. D. CLARKE. 1960. Effects of C02 on mi- activity of extracted mitochondria. The accelerated decline in tochondrial enzymes from Ricinus. Biochem. J. 76: 216-220. 21. RoIAN-I, R. J., I. K. Yr, AND L. K. FISHER. 1969. Isolation of tightly coupled capacity to oxidize succinate or NADH with time suggests that mitochondria from acidic plant tissues. Plant Physiol. 44: 311-312. mitochondrial damage was occurring. Furthermore, the intensi- 22. SARKISSIAN, I. V. AND H. K. SRIVASTAVA. 1968. On methods of isolation of ac- fication of this decline rather than a lessening or reversal of it tive, tightly-coupled mitochondria of wheat seedlings. Plant Physiol. 43: when fruit were transferred to air indicates that the damage 1406-1410. 23. T. DE. 1804. Recherclies Chiimiques stir la V-egetation. V. was irreparable. Noteworthy, however, was the sparing of the SAUlSSURE, Ny Ion. Paris. C02-sensitivity during this decline in activity. 24. SMtAGI-LA. J. 'l.. AV. J. BRAMLAGE, R. A. SOUTHWICK, AND H. V.IMARSH, JR. Prolonged exposure to CO2 reduced oxidative phosphoryla- 1968. Effects of watercore on respiration and ositochondrial activity in Ri- tion during much of the storage period. While CO2 clearly did chared Delicious apples. Proc. Amer. Soc. Hort. Sci. 93: 753-761. not act as a strong uncoupler of phosphorylation, the observed 25. SOUTHARD, J. H. 1970. Skeletal muscle mitochondrial hexokinase: character- istics and possible metabolic roles. Ph.D. of effect could have been consequential, reducing the energy level thesis. University Massachu- within the cell. Atkinson (1) has discussed the possible regula- setts, Amherst. 26. THOMAS,'M. 1925. The controlling influence of carbon dioxide. V. A quantita- tory mechanisms whereby a drop in the cellular energy level tive study of the production of ethyl alcohol and acetaldehyde by cells of would stimulate sugar utilization. In our study, we did find the higher plants in relation to the concentration of oxygen and carbondi- evidence of accelerated sugar utilization in the apple main- oxide. Biochem. J. 19: 927-947. 27. W. R. H. BURRIS, AND J. F. STAUFFER. Manometric Tech- tained in 12% CO2 and 3 % 02. to be presented in a later paper. UMBREIT, W., 1957. niques. Burgess Publishing Co.,Minneapolis. pp. 18-27. If despite accelerated sugar utilization the cellular energy re- 28. WILLIA'MS, M4. W. AND 'M. E. PATTERSON. 1964. Nonvolatile organic acids and mained at a reduced level, structural changes within the cell core breakdown of Bartlett pears. Agr. Food Chem. 12: 80-83. or mitochondria or both might occur and lead to progressive 29. WISKICH, J. T. 1966. Respiratory control by isolated apple mitocisondria. Na- loss of mitochondrial activity. ture 212: 641-642.