STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES

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1 STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES XVIII. THE EFFECT OF OXYGEN ON STARCH FORMATION AND DISSOLUTION IN POTATOES BY J. BARKER The Botany School, University of Cambridge {Received 30 July 1964) SUMMARY Earher evidence that starch is not formed from sugar in plant tissues in the absence of oxygen was confirmed for potatoes. The failure to form starch is attributed to a deficiency of glucosei-phosphate in nitrogen. As in other plant tissues the dissolution of starch to sugar also appeared to be retarded in potatoes in the absence of oxygen. This observation would be explained if starch is degraded by phosphorylase to glucose-1-phosphate, which is then converted to sucrose before partial inversion to hexose; the rate of formation of sucrose in nitrogen would be retarded by lack of uridine triphosphate. INTRODUCTION In leaves starch is either not formed from sugar or is formed only in traces in the absence of oxygen (Winkler, 1898; Phillis and Mason, 1937; Porter, 1953). The exclusion of oxygen also retards the formation of sugar from starch in leaves and other tissues (Spoehr and Milner, 1939; Porter, 1953). Our work indicates that both the formation and degradation of starch in potatoes are retarded by excluding oxygen. Possible explanations of these effects are given. Because of the high and variable amount of starch in potatoes, the starch content was not determined in our work. We assume, however, that when sugar content increases, the sugar is formed from starch; also that when sugar decreases this compound is partly converted to starch. METHODS The methods used were essentially those already described (Barker and Saifi, 1952). The main stock of potatoes, variety King Edward VII, was again stored at 10" C until required. For studying the infiuence of atmospheres either of nitrogen or of mixtures of oxygen and nitrogen, separate samples, each of from eighteen to thirty potatoes, depending on the particular experiment, were placed in sealed jars, through which passed a current, at the rate of about 2 litres per hour of either C02-free air, nitrogen or other gas as desired. The samples could thus be held for periods of weeks in the appropriate atmosphere. The total sugar content (sucrose+hexose) was determined using frozen potato powder and the procedure of Barker and Saifi (1952).

2 202 J. BARKER RESULTS Experiments i and 2 Experiment i. Stock A; stored 4 months at 1 C; to 10 C at o days, i.e. 11 July 1950; into nitrogen after 3 days at 10 C (Figs, i and 2) Total sugar content (Fig. i). After 4 months at 1 C the total sugar content (i.e. sugar) amounted to nearly 5 %. After change to 10 C sugar decreased to about 3.4 % in 15 days. After transfer to nitrogen at 3 days sugar appeared to decrease initially, as in air, but from 7 days there was little decrease. CO2 output (Fig. 2). The initial high rate of CO2 output at 10 C was followed by a decrease. In nitrogen the rate decreased by some 60 % of the original rate in air. t! Fig. I. Experiment i. Changes in content of total sugar at io C either in air ( x) or in nitrogen (O) after 3 days at io C. Arrows show time of change to io C and to nitrogen. Experiment 2. Stock A; stored 7 months at 1 C; to 10 C at o days, i.e. 6 November 1950; into nitrogen after 11 days at 10 C (Fig. 3) Sugar (Fig. 3). The decrease of sugar in air at 10 C was retarded by transfer to nitrogen. CO2 output. The rate of CO2 output in nitrogen was again slower than in air. Comment on experiments i and 2 In experiments i and 2 the potatoes had been allowed to sweeten fully at 1 C before transfer to 10 C, at which temperature de-sweetening was in progress when oxygen was excluded. Previous work has shown that in de-sweetening in air sugar is mainly condensed to starch, only a part of the decrease in sugar being due to consumption in respiration (MuUer-Thurgau, 1882). Our object was to ascertain the effect of excluding oxygen on the rates of de-sweetening and of condensation to starch. As mentioned above, changes

3 Oxygen and starch in potatoes 203 Fig. 2. Experiment i. Changes in rate of CO2 output at 10 C either in air ( nitrogen ( ) after 3 days at 10 C. Arrows show time of change to 10 nitrogen. ) or in and to 6-0 gi I s I ^ Fig. 3. Experiment 2. Changes in content of total sugar at 10 C either in air (x) or in nitrogen (o) after 11 days at 10'"' C. Arrows show time of change to 10' C and to nitrogen.

4 204 J. BARKER in starch content were not determined; any loss of sugar, not accounted for by respiration is assumed to be due to starch formation. For Tables i and 2 the sugar consumption by respiration in air was calculated assuming a respiratory quotient of i.o. The consumption of sugar by fermentation in nitrogen was calculated as the sugar equivalent of the CO2 output in nitrogen multiplied by three; a factor of three was given by the ratio CO2 + lactate in nitrogen/co2 in air in experiment i. Column 4 (Table i) lists values for the observed decrease in sugar in air and in nitrogen in experiments i and 2. Nitrogen retarded the decrease in sugar. Since, however, sugar is probably also being used in respiration or fermentation, column 5 gives calculated values for sugar consumption in these processes and column 6 lists the difference (column 4 column 5), which represents the amount of sugar converted to reserves, assumed here to be starch. In experiment i no sugar was converted to starch in nitrogen. In experiment 2 column 6 has a large negative value in nitrogen, indicating not only that no sugar was converted to starch but that a large amount of sugar was produced from starch for consumption in fermentation. Table i. Observed and calculated changes in sugar in air and in nitrogen {values expressed as % sugar per 100 g fresh weight) Experiment Period Atmosphere Observed Sugar con- Sugar (days) decrease sumption in converted in sugar respiration or to starch fermentation (4 5) Air Nitrogen Air Nitrogen Experiments 3 and 4 Experiment 3. Stock B; immature potatoes lifted and to 1 C,T, August 1949; into nitrogen after 9 days at 1 C (Fig. 4) Sugar (Fig. 4). Sugar increased in air at 1 C throughout the experiment. After 9 days at 1 C, exclusion of oxygen retarded the rise in sugar, particularly after 10 days in nitrogen. COo output. Nitrogen again decreased the rate of CO2 output. Experiment 4.. Stock C; immature potatoes lifted and to 1 C, i^ August ig^o; into nitrogen after 19 days at 1 C (Fig. 5) Sugar (Fig. 5). Sugar increased at 1 C throughout the experiment. Exclusion of oxygen retarded the rise in sugar, particularly after 13 days in nitrogen. CO2 output. Again the CO2 output decreased in nitrogen. Comment on experiments 3 and 4 In contrast with the de-sweetening of experiments i and 2, the potatoes of experiments 3 and 4 were sweetening at the low temperature; the bulk of the sugar accumulated is known to be derived from starch (Muller-Thurgau, 1882). The intention was to ascertain the influence of excluding oxygen on the rate of increase in sugar and on the calculated rate of production of sugar from reserves, assumed to be starch. In experiments 3 and 4 transfer to nitrogen greatly retarded both the observed increase

5 Oxygen and starch in potatoes en Fig. 4. Experiment 3. Changes in content of total sugar at 10" C either in air ( ) or in nitrogen ( :) after 9 days at -i" C. Arrows show time of change to -i" C and to nitrogen Fig. 5. Experiment 4. Changes in content of total sugar at 1 C either in air ( x) or in nitrogen (O) after 19 days at 1 C. Arrows show time of change to i'" C and to nitrogen.

6 2O6 J. BARKER in sugar (Table 2, column 4) and the calculated value for sugar formed from starch (column 6). Column 7 lists the reduction, in nitrogen, in the rate of formation of sugar from starch expressed as a percentage of the rate in air. Table 2. Observed and calculated changes in sugar in air and in nitrogen (values expressed as % sugar per 100 g fresh weight) I Experiment Period (days) Atmosphere Air Nitrogen Air Nitrogen Air Nitrogen Air Nitrogen 4 Observed increase in sugar Sugar consumption in respiration or fermentation O-33 6 Sugar formed from starch (4 and 5) I.II Reduction, in nitrogen, of rate of sugar from starch (%) Experiment 5 Experiment 5. Stock D; stored at 10 C for 6 months and transferred to 1 C on 20 March 1935; either in air, in 60 % oxygen, or in 5 % oxygen (Figs. 6 and 7) Sugar (Fig. 6). In air at 1 C sugar increased to a high value of about 4.5 %. In 60 % oxygen sugar rose to about 5.1 % and in 5 % oxygen to 4.1 %. CO2 output (Fig. 7). In air CO2 output increased and tben decreased as is cbaracteristic of 1 C (Barker, 1933). The rate was higher in 60 % and lower in 5 % oxygen than in air. Sugar and CO2 output also increased faster initially at 1 C in 100% oxygen than in air (Barker and Mapson, 1955). DISCUSSION Possible enzymic mechanisms In vitro studies (Whelan, 1961) indicate that starch may be hydrolysed to sugar in plants by amylases and maltase or be transformed by starcb phosphorylase into glucose-1- phosphate; this ester may then be converted either into free hexose sugars or to sucrose phosphate followed by partial inversion to hexoses (Fig. 8). Starch may be synthesized from glucose-1-phosphate by starch phosphorylase or possibly from uridine diphosphate glucose by amylose synthetase (Whelan, 1961). Attempts to determine which enzymic mechanisms are active in potatoes in vivo have met with little success. Arreguin-Lozano and Bonner (1949) reported that an inhibitor of starch phosphorylase accumulated in potatoes at high temperatures, the inhibitor disappearing at low temperatures. This evidence may indicate that sweetening at low temperatures is due to phosphorylase action but Porter and Rees (1954) could not confirm tbe observation of Arreguin-Lozano and Bonner. Potato extracts contain little, if any P-amylase activity (Whelan, 1963, personal communication) and Porter and Rees (1954) found no difference in content of short chain dextrins in extracts from potatoes sweetening rapidly at 0 C and from potatoes with stable sugar at 25 C; these observations may mean that amylases do not participate in sweetening. The changes in various phosphate esters during

7 Oxygen and starch in potatoes 207 sweetening at low temperatures were determined by Arreguin-Lozano and Bonner (1949) and by Samotus (i960); the alteration of the key ester, glucose-1-phosphate was not, however, estimated by a reliable technique gi % Fig. 6. Experiment 5. Changes in content of total sugar at i'c either in air ( ; ') in 60% oxygen (c) or in 5% oxygen (A). Arrows show time of change to 1 C and to 60% or 5% oxygen Fig. 7. Experiment 5. Changes in rate of CO2 output at 1 ' C either ii in air (- -), 60% oxygen ( ) or in 5 % oxygen ( ). Arrows show time of change to 1 C and to 60% or 5 % oxygen. Exclusion of oxygen and formation of starch As shown earlier with leaves, excluding oxygen prevented formation of starch in our experiments. Since phosphorylase is active in vitro in the absence of oxygen

8 2o8 J. BARKER (Porter, 1953), this effect may be attributed to a decreased rate of formation of glucose-iphosphate in nitrogen due to shortage of adenosine triphosphate. But it is not known whether glucose-1-phosphate decreases when oxygen is excluded. Exclusion of oxygen and breakdown of starch Excluding oxygen retarded the formation of sugar from starch by from 57 to 87 % of the rate in air (Table 2). After our work was completed, we became aware that this effect had been previously observed in other tissues. Thus Spoehr and Milner (1939) report that excluding oxygen retarded tbe loss of starch from leaves by % of the rate in air. Porter (1953) comments 'to maintain degradation in vivo it is presumably necessary to remove the primary products of breakdown by an oxygen dependent step'. Considering now the phosphorylase system, the rate of breakdown of starch would be expected to be faster in nitrogen since the concentration of inorganic phosphate should be higher than in air (Rowan, Seaman and Turner, 1956; Lynen, Hartman, Netter and Scheugraf, 1959; Beevers, 1961), but this probability does not mean that sugar formation would necessarily be faster in nitrogen. Two possible routes of formation of sugar may be distinguished. In route i, glucose-1-phosphate would be converted to uridine dipbosphate starch uridine triphosphate 7 glucose-1-phosphate;=^uridine diphosphate ^sucrose phosphate glucose glucoses glucose-6-phosphate /'"^ sucrose + phosphaie fructose-6-phosphate glucose + fructose fructose Fig. 8. Possible mechanisms of formation of sucrose and hexose from starch via starch phosphor>'lase. For simplicity the synthesis of sucrose from uridine diphosphate glucose and tructose is not shown. glucose and this to sucrose, which might be partly inverted to hexoses (Eig. 8). In route 2, either glucose-1-phosphate, or glucose- or fructose-6-phosphates, formed from glucose- I-phosphate, would be bydrolysed to free hexoses (Eig. 8). If then in air sugar was formed mainly by route i, a large retardation of sugar formation, of the order observed above, would be expected to occur on excluding oxygen, since tbe supply of uridine triphosphate would decrease greatly. Moreover, our observations showed that after longer periods in nitrogen the retardation of sugar formation was greater (Table 2). This finding would be in accord with the continued slow operation initially in nitrogen of route i, since after longer periods the fermentation rate was also slower (Table 2). Alternatively, the activity of starch phosphorylase might decrease after a longer time in nitrogen (see p. 209). Our observation that excluding oxygen retarded sugar formation may thus be evidence that the primary route of starch dissolution is to sucrose. A test of tbis hypothesis would be wbetber glucose-1-phosphate accumulated, and uridine diphosphate glucose decreased, in nitrogen. Porter and Bird (1962) argue from changes in specific activity of i^c in tobacco leaves that starch dissolution occurs by route i (Eig. 8) but the evidence does not exclude route 2.

9 Oxygen and starch in potatoes 209 Moreover, if our calculated values for starch to sugar in nitrogen are correct (Tables i and 2), a substantial breakdown of polysaccharide still occurs in nitrogen. The dissolution of a considerable quantity of starch in leaves held in nitrogen was earlier shown by actual determination of starch loss (Spoehr and Milner, 1939). This dissolution must be attributed either to amylase activity or to phosphorylase action by either routes i or 2. Since sucrose always decreases in potatoes in nitrogen and the net increase in sugar in nitrogen, when it occurs (Table 2), is in hexoses, route i is probably not then the main pathway. Oxygen concentration and extent of sweetening In experiment 5 sweetening was greater in 60 % oxygen than in air and greater in air than in 5 % oxygen (Fig. 6). Since the respiration decreased in the same order as sugar content (Fig. 7), these observations would be consistent with the theory suggested above of control of sweetening by rate of supply of uridine triphosphate. ACKNOWLEDGMENTS This work was completed while I was on the staff of the Low Temperature Research Station of the Department of Scientific and Industrial Research. I wish to thank Professor C. S. Hanes, F.R.S., and Dr. T. Solomos for valuable suggestions and Mr. P. Curtis, Mr. H. A. F. Jackson, Miss C. Lambert, Mr. F. Lenton and Mr. H. Moore for their help. I wish to thank Frederick Hiam Ltd., Cambridge, from whose farms the potatoes were obtained. REFERENCES ARREGUIN-LOZANO, B. & BONNER, J. (1949). Experiments on sucrose formation by potato tubers as influenced by temperature. Plant PhysioL, 24, 720. B.\RKER, J. (1933). The effect of temperature-history on the respiration/sugar relation. Proc. i?..soc, B112, 316. BARKER, J. & SAIFI, A. F. EL (1952). Experimental studies of the formation of carbon dioxide, lactic acid and other products in potato tubers under anaerobic conditions. Proc. R. Soc, B140, 362. B.\RKER, J. & MAPSON, L. W. (195s). Experimental studies with potato tubers of an inhibition of the respiration and of a 'block' in the tricarboxylic acid cycle induced by 'oxygen poisoning'. Proc. R. Soc, B BEEVERS, H. (1961). Respiratory Metabolism in Plants. Row, Peterson, New York. LYNEN, F., HARTMAN, G., NETTER, K. F. & SCHEUGRAF, S. (1959). CIBA Foundation Symposium on The Regulation of Cell Metabolism. Churchill, London. MULLER-THURGAU, H. (1882). Uber die Natur des in sussen Kartoffein sich vorhndenen Zuchers. Landw. Jb., II, 751. PHILLIS, E. & M.^ON, T. G. (1937). On the effects of light and of oxygen on the uptake of sugar by the foliage leaf. Ann. Bot., N.s., i, 231. PORTER, H. K. (1953). Starch synthesis and degradation in vivo. Biochem. Soc. Symp., 11, 27. PORTER, H. K. & BIRD, I. F. (1962). Assimilation and respiration by tobacco lead tissue. A quantitative study using ^*C. Indian jf. Plant PhysioL, 5, 5. PORTER, H. K. & REES, W. R. (1954). Some effects of ethanol extracts of potatoes on the activity of a phosphorylase preparation. Plant PhysioL, 29, 514. ROWAN, K. S., SEAMAN, D. E. & TURNER, J. S. (1956). Phosphorylation as a possible factor in the Pasteur effect in plants. Nature, Lond., 177, 333. SAMOTUS, B. (i960). The role of phosphorus in biochemical transformation of starch in potato tuber during the vegetation period and different temperatures of storage. Report to Rockefeller Foundation (unpublished). SPOEHR, H. A. & MILNER, H. (1939). Starch dissolution and amylolytic activity in leaves. Proc Am.phil. Soc, 81, 37. WHELAN, W. J. (1961). Recent advances in the biochemistry of glycogen and starch. Nature, Lond., 190, 954. WiNKLER, H. (1898). Untersuchungen uber die Starkebildung in den verschiedenartigen Chromatophoren. Jb. wiss. Bot., 32, 525. C NP.

[ II ] BY J. BARKER. Low Temperature Research Station, University of Cambridge {Received 12 February 1949) (With 6 figures in the text)

[ II ] BY J. BARKER. Low Temperature Research Station, University of Cambridge {Received 12 February 1949) (With 6 figures in the text) [ II ] THE ASCORBIC ACID CONTENT OF POTATO TUBERS. I. THE RELATION BETWEEN ASCORBIC ACID AND THE SUGAR CONTENT, AS INFLUENCED BY THE MATURITY AT LIFTING AND BY STORAGE BY J. BARKER Low Temperature Research