[ 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 Station, University of Cambridge {Received 12 February 1949) (With 6 figures in the text) I. INTRODUCTION No detailed information is available concerning the mechanism whereby ascorbic acid is formed in plant tissues, but Ray (1934) showed that, with germinating pea seeds, the addition of certain sugars e.g. glucose, fructose, sucrose and mannose increased the formation of ascorbic acid. It has long been known that the sugar content of potato tubers can be varied widely by appropriate changes of the temperature of storage. Using a slight modification of the indophenol technique, as described by Pett (1936), a preliminary experiment was carried out in 1936. Potatoes, variety King Edward VII, of low sugar content were transferred from 10 to 1 C. The total sugar and the ascorbic acid contents both increased markedly during the first 18 days at 1 C.; after about 20 days, however, the ascorbic acid content* began to fall and, in spite of a further large increase of the sugar content, the fall continued until a low value was reached. A similar experiment, with the same variety of potatoes but with a different stock, showed no rise of ascorbic acid at 1 C, although the sugar content increased as usual to a high value. Following these preliminary observations, a comprehensive investigation of the relation between ascorbic acid and the sugar content was initiated. The first objective of this study was to establish the magnitude and direction of the changes in the ascorbic acid content of potato tubers under a variety of conditions. The second aim was to ascertain whether any one of the major changes in the content of ascorbic acid was associated with parallel drifts in sugar content. 2. METHODS The potatoes, variety King Edward VII, were taken from good quality commercial crops, grown on medium loam soils near Cambridge, f The various stocks of potatoes required were dug by hand, the liftings all being made from a small area of the field. Small tubers of between 25 and 35 g. in weight, which had already proved satisfactory material for physiological studies, were adopted as the standard unit. For each determination of ascorbic acid content two samples, each of six potatoes, were taken. In preparing each sample for analysis, median longitudinal slices were * Here and throughout the paper 'ascorbic acid content' is employed for the amount per 100 g. fresh weight of potato tissue. f I wish to thank Chivers and Sons Ltd., Histon, from whose farms the potatoes were obtained.
12 J. BARKER collectively extracted with an equal volume/weight of 5 % trichloracetic acid. The extracc was titrated with 2:6-dichlorophenolindophenol before and after treatment with HgS. Since the increase in reducing value following treatment with HgS rarely exceeded 10%, only the figures obtained after reduction are considered here; for brevity these values are termed ascorbic acid throughout the paper, although they actually represent total ascorbic acid (i.e. ascorbic-i-dehydroascorbic acid). In a later paper it is hoped to analyse the relation between ascorbic and dehydroascorbic acid under different conditions. For simplicity only the mean values for ascorbic acid for each pair of samples are recorded in the figures. The values for sugar content appeared to be more variable than those for ascorbic acid, and the number of standard-size potatoes, taken for sugar analysis, was increased to twenty tubers per sample. The analyses of ascorbic acid and sugar could not, in any case, be made on the same potatoes, since the former were carried out in London and the latter in Cambridge. Every precaution was taken to ensure that the samples used for these two determinations were comparable in origin and in storage treatment. For the samples posted to London the period of i day between removal from storage at 10 C. and analysis was inevitably at an uncontrolled temperature. Preliminary trials showed that rough handling of potatoes, such as might occur in postal transit, did not appreciably affect the ascorbic acid content. The technique used for the analysis of the sugar content has already been described (Barker, 1936). 3. CHANGES OF ASCORBIC ACID CONTENT DURING GROWTH AND DEVELOPMENT ON THE PLANT In each of the three seasons 1937, 1938 and 1939 stocks of potato tubers were lifted at approximately 3-weekly intervals, beginning either at the end of June or about 20 July and continuing until the crop was gathered commercially. At the earlier diggings the skin was not set, the tubers being the more immature, as judged by the ' slipping' of the skin, the earlier the date of lifting. At each lifting samples were taken for immediate analysis; the remainder were placed in damp peat moss at 10 C, samples being removed for analysis at appropriate intervals during the ensuing 200-400 days. The sprouts, which developed after some months in storage, were removed when they were about ^ in. in length. The ascorbic acid content of the samples, analysed immediately after each lifting, are plotted against the date of analysis in Fig. i see period June to October; interrupted lines have been drawn linking the points for each year; the date of the final or commercial lifting is marked by a vertical arrow. In each season the ascorbic acid content was higher at the second than at the first lifting; a maximum content appeared to be reached in August or in early September. In both the 1937 and the 1938 seasons there was a marked fall of the ascorbic acid content during September. This fall was not so pronounced in 1939. Thus, during the growth and development of the potatoes up to full maturity in the soil the ascorbic acid content, determined for each lifting by analysing tubers of the standard size shortly after digging, first rose to a maximum and then fell; this confirms the observations made by Smith & Gillies (1940) and by Lampitt, Baker & Parkinson (1945). There were, however, marked differences between the results for the three seasons, both in the magnitudes of the ascorbic acid content, attained at any given date, and in the forms of the drift with time of the ascorbic acid content in each season. For example, the maximum
Ascorbic acid in potatoes 13 value observed in 1937 was 44-2 mg./ioo g. fresh weight as against a maximum figure of 35-2 in 1939. Moreover, the data indicated that the ascorbic acid content began to fall at about the middle of August in 1938, but not until early September in 1937. These and other differences, shown in Fig. i, must be presumed to be due to differences in the seed used or in the soil or climatic conditions in the three seasons. June Fig. I. Ascorbic acid content of successive liftings in the 1937, 1938 and 1939 seasons; points for each season linked by interrupted line and date of final lifting marked by vertical arrow. Also decrease in ascorbic acid content of final lifting during storage at 10 C. ; continuous lines through points for each season, x, 1937; O, 1938;, 1939. One factor which may have determined, in part, the fall in the ascorbic acid content was the death of the haulm, which began to occur about a month before the final date of lifting. The decrease in ascorbic acid during this period may thus be partly due to a gradual cessation of the supply to the tubers either of ascorbic acid or of a precursor of the vitamin. No critical investigation of this interpretation was made, but in an examination of tubers lifted on 20 September 1938, samples from plants with green haulm contained 32-2 mg. ascorbic acid per 100 g. fresh weight compared with 25-6 mg. for samples from plants with dead haulm. The early fall of the ascorbic acid content in 1938 (Fig. i) cannot, however, be explained on this basis, since the haulm began to die at about the same date in each season. It is important to decide whether the initial values, recorded for each lifting in Fig. i, were seriously affected by the delay of one day, which necessarily elapsed between the time of digging and the analysis. A careful survey of the data for the decrease of the ascorbic acid content (in mg./ioo g. fresh weight) in storage at 10 C. following lifting
14 J. BARKER (see next section) justifies the conclusion that, while the actual values of the points o Fig. I would presumably have been higher, had there been no delay before analysis, the forms of the curves would not have been seriously affected. As already noted, the data in Fig. i were obtained by analyses of a single standard size of tuber. Comparisons showed, however, that the ascorbic acid content (in the units above mentioned) appeared to be little affected by the size of the tuber, either immediately after lifting or during storage. Since a similar result has been reported in a number of lnvestiga- Fig. 2. Decrease in ascorbic acid content in storage at io C. in stocks lifted at four different dates in 1937. Vertical arrows show dates of lifting. Initial points for the four stocks linked by interrupted line; continuous lines through points for each individual stock. O, first lifting; x, second lifting;, third lifting; A, fourth lifting. tions (Esselen, Lyons & Fellers, 1942, and Dove, Murphy & Akeley, 1943), the general trends recorded in Fig. i can probably be safely accepted as indicating the drifts which would have occurred in tubers of all sizes. 4. CHANGES IN ASCORBIC ACID CONTENT DURING THE STORAGE OE IMMATURE AND MATURE POTATOES AT IO C. As recorded in the previous section, stocks of potatoes were lifted on four separate occasions during the growth of the crop in the 1937 season (Fig. i). On each occasion, in addition to the analysis made immediately after lifting, further determinations were made at appropriate intervals during storage at 10 C. In plotting the results in Fig. 2, the initial points for each stock have been linked, as in Fig. i, by an interrupted line; in
Ascorbic acid in potatoes 15 contrast the drift of ascorbic acid with time at 10 C. in the samples of each lifting is indicated by a continuous line; the date of each lifting is shown by a vertical arrow. In each stock there was a long-continued fall of the ascorbic acid content during storage at 10 C. (Fig. 2). Both the most rapid and the largest fall occurred in the first lifting namely that on 20 July. The stocks dug on 8 August and 9 September showed rates of fall of ascorbic acid content intermediate between the first and the final liftings. In February, 1938, when the tubers ofthe first lifting had been in storage for 200 days, the ascorbic acid content was appreciably below that of the final lifting; the values for the second and third liftings were intermediate (Fig. 2). The metabolic condition of the tuber, when lifted, must thus exert a very marked effect on the rate of disappearance of ascorbic acid in storage. A further point of interest was that after 150-200 days in storage, the values for the first, second and third liftings showed little or no further fall with continued storage. Rapid losses of the ascorbic acid in immature tubers were also observed in 1938 and 1939 (data not recorded here). The drifts of the ascorbic acid content in storage at ro C. for mature tubers of the final lifting for each of the three seasons, 1937, 1938 and 1939 are recorded in Fig. i; the initial point of each series is marked with a vertical arrow. Although the rate of loss of ascorbic acid content was not as rapid as in potatoes lifted immature, nevertheless, the value fell by 50% in from 3 to 4 months; the rate of fall became progressively slower the longer the potatoes were held in storage. The three stocks showed parallel decreases in storage. In early October the value for 1937 was roughly 50% above that for 1938 and this difference was still evident in the following May. 5. RELATION BETWEEN ASCORBIC ACID AND THE SUGAR CONTENT DURING STORAGE AT IO C The sugar content was determined, both immediately following lifting and at intervals during subsequent storage at 10 C, in samples from each of the four stocks lifted during the 1937 season. The hexose and sucrose contents have been plotted against time in Fig. 3 A and B; as in Fig. 2, which gives the corresponding data for ascorbic acid, the initial points for the four stocks have been linked by an interrupted line; continuous lines have been drawn through the successive points for each stock. In contrast to ascorbic acid, which increased from the first to the third lifting (Fig. 2), the initial values of both hexose and sucrose fell with advancing maturity (Fig. 3). During storage at 10 C, the hexose content rose initially in each series (Fig. 3 A). Thus, for each stock, the drifts of hexose and of ascorbic acid were in opposed directions for at least 30 days in storage at 10 C. That this inverse relation applied to both the hexose sugars present was shown by separate estimations of fructose and glucose. Fach of these sugars increased follovi^ing lifting, and the forms of their drifts in storage were similar to those shown for the combined content of hexoses in Fig. 3 A. For the sucrose content (Fig. 3B), the relation was quite different. In the first and second liftings the sucrose content was high at the time of digging i.e. 0-92 and 0-83 % respectively; a marked fall began immediately and continued, though becoming progressively slower, for at least 100 days. These falls of sucrose resembled closely the corresponding decreases of ascorbic acid in the first and second liftings (Fig. 2). The initial sucrose content of the third and final liftings was lower than that of the first and second liftings, and the initial change in storage was a small increase of the sucrose content. This
i6 J. BARKER increase, however, lasted only for about 20 days in the third lifting, and for 10 days in the final lifting, prior to prolonged falls of sucrose in both liftings. Apart from the initial temporary increase, there was thus again a parallelism between the drifts of sucrose and of ascorbic acid (Fig. 2). ~ 1-0 50 100 Days 150 200 250 300 o 075 0-5 A, Hexose I 0-25 ^-c. r 1 0-75 ^0-50 B. Sucrose!0-25 Aug. Sept. Oct Nov. Dec. Jan. Feb. Mar. Apr. May _L Fig. 3. Changes in hexose and sucrose contents during storage at 10 C. in stocks lifted at four different dates in 1937. Vertical arrows show dates of lifting. Initial points for the four stocks linked by interrupted line; continuous lines through points for each individual stock. O, first lifting; x, second lifting-, third lifting; A, fourth lifting. In the 1938 season, sugar data were obtained only for the first and third liftings. In the first lifting the hexose content was again low, and a marked rise occurred on storage at 10 C. In contrast the sucrose was initially high, 07%, and continued to fall in storage
Ascorbic acid in potatoes 17 for at least 190 days. For the third lifting, the hexose was again low initially and increased in storage for at least 30 days; the sucrose showed the usual prolonged fall. Thus, as in 1937) the drifts of the hexose sugars and of the ascorbic acid (see p. 15) were in opposite directions for at least 30 days in storage; sucrose and ascorbic acid again showed a marked resemblance in their drifts. In 1939 analyses of the sugar content were made only for the third and final liftings. The drifts of hexose and ascorbic acid were again in opposed directions, while sucrose and ascorbic acid both decreased in storage. o 1st lifting 1937 X 2nd lifting 1937 3rd lifting 1937 A4th lifting 1937 o 1st lifting 1938 3rd lifting 1938 X 2nd lifting 1939 A 4th lifting 1939 0-5 0-75 0 0-25 0 0-25 0-5 0 Sucrose content (as percentage of fresh weight) 0-25 0-5 075 Fig. 4. Relation between ascorbic acid and sucrose contents during storage at 10 C. A: O, first lifting 1937; X, second lifting, 1937. B: third lifting, 1937; A, fourth lifting, 1937. C: O, first lifting, 1938;, third lifting, 1938. D: x second lifting, 1939; A, fourth lifting, 1939. Final point of each series is marked with duration of storage in days. Relation between ascorbic acid and sucrose. To examine more closely the form of the relation between the contents of ascorbic acid and sucrose, the data of Figs. 2 and 3 B have been replotted in Fig. 4A, B; each stock has been treated separately, the ascorbic acid values being plotted against the appropriate sucrose contents. The corresponding data for the 1938 and 1939 seasons are shown in Fig. 4C, D. The ascorbic acid values used in Fig. 4 are those actually determined; where no corresponding figure for sucrose is available at the precise date of the ascorbic acid analysis, a value has been interpolated from the time-drifts. In Fig. 4 A-D there are thus eight separate sets of data for the relation between ascorbic acid and sucrose, and each of these sets was obtained during the fall of ascorbic acid in storage at 10 C. in a single stock following lifting. For each stock the highest ascorbic acid value, shown in Fig. 4, is derived from the analysis made immediately following lifting; the time sequence of the points is thus towards the origin, and the point nearest the origin i.e. lowest in ascorbic acid represents the sample stored longest at 10 C; for convenience, the actual duration of storage for the final sample of each series is indicated in Fig. 4. With one exception, each separate set of data in Fig. 4 shows a fair approximation to New Phytol. 49, 1 «
i8 J. BARKER a linear relation through the origin, with a tendency to curve away from the ordinate axis in higher sucrose contents. The data for the fourth lifting in 1939 (Fig. 4D) do not appear to conform with this relation, but the fall in ascorbic acid, exhibited by the three points comprising the series, is not sufficiently large to show whether this stock was really aberrant or not. Departure from the general relation should, however, be noted in three of the eight series of related data: namely in Fig. 4A (open circles in low sucrose content for the first lifting of 1937), and in Fig. 4B (solid dot m high sucrose content for the third lifting of 1937, and triangle in low sucrose content for the fourth lifting of 1937)- While these anomalous values no doubt indicate the occasional intervention of additional factors (see p. 21) the data of Fig. 4 justify the general conclusion that, for any one stock of potatoes under the given conditions, a close correlation exists between the ascorbic acid content and the sucrose concentration. Another feature of Fig. 4 is the wide variation in what may be termed the ' pitch' of the ascorbic acid/sucrose relation. For example, in Fig. 4A, the values for ascorbic acid content in the second lifting (crosses in Fig. 4A) are markedly higher for a given sucrose content than those for the first lifting (open circles in Fig. 4A). In order to assess quantitatively the pitches of the various curves in Fig. 4 we have derived from each curve an ascorbic acid/sucrose factor, this being the ascorbic acid value corresponding to a content of 0-2% sucrose. The eight factors so obtained have been plotted in Fig. 5 A against the date at which the particular stock of potatoes was lifted, i.e. the ascorbic acid/sucrose factors for stocks lifted in the three different seasons 1937, 1938 and 1939, are plotted to a common time-scale. The distribution of the eight points in Fig. 5 A suggests strongly that the variation in the pitches of the ascorbic acid/sucrose curves in Fig. 4 is not haphazard, but that the ascorbic acid/sucrose factor is lowest in the stock from the first lifting and increases progressively to the final lifting. It was noted earlier that while the initial values of ascorbic acid content increase from the first to the third lifting and then decrease (Fig. 2), the corresponding sucrose contents fall markedly with advancing maturity (Fig. 3B). This divergence is refiected in the increase in the ascorbic acid/sucrose factor with maturity shown in Fig. 5 A. The changes, already noted above, in the relative balance of sucrose and the hexoses during storage at 10 C. (Fig. 3) are conveniently recorded by calculating sucrose/hexose quotients and plotting the drifts of these against time (Fig. 6). Comparing Fig. 6 with Fig. 2 for the corresponding changes in ascorbic acid, the marked decreases in both functions following lifting are evident. The actual forms of the decreases have little in common; thus the sucrose/hexose quotients show an extremely rapid initial fall, with only a slight further decrease after about 20 days in storage; this behaviour contrasts with the long continued fall of the ascorbic acid. After some months in storage, however, the values for both the ascorbic acid content and the sucrose/hexose quotient are lower for the first and second liftings than for the third and fourth liftings. It was, therefore, decided to survey the sucrose/hexose quotients of the eight stocks considered above. For this purpose the values of the sucrose/hexose quotient at 0-2% sucrose termed adjusted quotient have been interpolated from the curves in Fig. 6 and the corresponding curves, not presented here, for the 1938 and 1939 data. The eight adjusted quotients are plotted in Fig. 5B against the date at which the particular stock of potatoes was lifted; the abscissa scale again represents a common time-scale for each of the seasons 1937, 1938 and 1939.
Ascorbic acid in potatoes 19 With the exception of one point namely the cross in Fig. 5B at quotient =1-5 and date 7 September the positions of the points in Fig. 5B indicate that the adjusted sucrose/hexose quotient increases progressively with advancing maturity at lifting. If now. Fig. 5 B is compared with Fig. 5 A, it is evident that the low ascorbic acid/sucrose factor of the most immature stock is correlated with a low adjusted sucrose/hexose quotient; similarly, the high ascorbic acid/sucrose quotient of the mature lifting is related to a high July Aug. Sept. Oct. Fig. 5. Effect of maturity at lifting on: A, ascorbic acid/sucrose factor; B, adjusted sucrose/hexose quotient. Each factor or quotient is plotted against the date at which the stock was lifted, x, 1937; O, 1938;, 1939- adjusted sucrose/hexose quotient. There is here a strong indication that variations in the ascorbic acid/sucrose factor may, under certain conditions, be associated with differences in the sucrose/hexose quotient. With reference to the three anomalous points in Fig. 4, already noted on p. 18, no explanation can be advanced for the marked departure shown by the open circles in low sucrose in Fig. 4A and by the solid dot in high sucrose in Fig. 4B; the latter sample had.
20 J. BARKER however, the very high sucrose/hexose quotient of 12-9 and during the initial perio in storage at 10 C. the sucrose content increased (Fig. 3B). The divergence of the open triangles in very low sucrose (Fig. 4B) may be a normal feature of the relation betvi^een ascorbic acid and sucrose after prolonged storage. Thus with potatoes dug when mature, the sucrose content usually increases following long storage at 10 C; further data are needed to show whether this increase is or is not accompanied by an increase of ascorbic acid. 12-5 - t I I 1 o I 1 1 O Fig. 6. Changes in sucrose/hexose quotient during storage at 10 C. in stocks lifted at four different dates in 1937. Vertical arrows show dates of lifting. Initial points for the four stocks linked by interrupted line; continuous lines through points for each individual stock. O, first lifting; x, second lifting;, third lifting; A, fourth lifting. May 6. DISCUSSION The data, given above, confirm the earlier evidence of OUiver (1938) that, immediately following lifting, immature potatoes are richer in ascorbic acid than mature potatoes and that the content decreases rapidly in storage. Similar results have been published by a number of other workers (Scheunert, Reschke & Kohlemann, 1937; Smith & Gillies, 1940). No serious attempt appears, however, to have been made to investigate the mechanism responsible for this fall. The evidence, presented above and summarized in Fig. 4, appears to justify the conclusion that, during the fall of ascorbic acid in storage at 10 C. following lifting, the
Ascorbic acid in potatoes 21 content of the vitamin is closely correlated with the content of sucrose. Moreover, the correlation is not confined to potato tubers, since a similar relation has been observed by Mapson (1948) in detached leaves. One interpretation of the significance ofthe correlation is that ascorbic acid and sucrose are formed from a common precursor. An alternative view is that both the formation of ascorbic acid and the synthesis of sucrose are favoured by similar metabolic conditions, i.e. the concentrations of the two constituents are separately determined by the rate of a third process. From work in progress it is clear, however, that the sucrose content is only one of several factors which appear to influence the ascorbic acid content of potato tubers. Not until the relation between ascorbic acid and these factors has been examined in a range of stocks of potatoes, subjected to various treatments, will it be possible to assess the significance of the observed correlation between ascorbic acid and sucrose. No attempt will thus be made at this stage to decide between the above and other possible interpretations of the mechanism underlying the observed correlation. As shown in Fig. 5 A, B, immaturity appears to be associated with a low ascorbic acid/sucrose factor and a low adjusted sucrose/hexose quotient, and maturity with a highei factor and a higher quotient. This suggests that the metabolic conditions, which favour the formation of ascorbic acid, are also conducive to the synthesis of sucrose. An increase in our knowledge of the latter process may thus assist the analysis of the former system. Earlier work has shown that, under certain well-defined conditions, the respiration of potato tubers is closely correlated with the content of sucrose (Barker, 1936). The observed relation between ascorbic acid and sucrose may thus reflect an interaction between the respiratory mechanism and the formation or loss of ascorbic acid. In the work of Ray (1934), fructose and sucrose appeared to be slightly more effective than glucose in increasing the ascorbic acid content of pea seedlings. These results were, however, based on the external addition of sugar solutions; no evidence was obtained of the changes in the internal concentrations of the different sugars. With cress seedlings Mapson, Cruickshank & Chen (1949) have found that the content of ascorbic acid is correlated with the concentration of hexose and that there is no correlation between ascorbic acid and sucrose. In view of the above results for the potato it would be interesting to know whether, in cress seedlings, respiration is more closely linked with the content of hexose than with that of sucrose. 7. SUMMARY The ascorbic acid content of potato tubers, variety King Edward VII, increased to a maximum and then decreased during their growth and development on the plant. The loss of ascorbic acid, in storage at 10 C. following lifting, was more rapid in potatoes, lifted immature, than in those dug when the haulm was dead. During storage at 10 C. the hexose content increased initially, but the sucrose content decreased; the relation between the falling ascorbic acid and sucrose contents was nearly linear. The metabohc significance of this correlation is discussed. The maturity of the tubers, when lifted, influenced both the relation between ascorbic acid and sucrose, during storage at 10 C., and the balance between sucrose and the hexose sugars.
22 J. BARKER I wish to thank Dr L. W. Mapson and Dr C. S. Hanes for their interest in the investigation. The sugar estimations were carried out by Mr H. A. F. Jackson and technical assistance was given by Mr A. E. Porter. The work described in this paper was carried out as part of the programme of the Food Investigation Organization of the Department of Scientific and Industrial Research. REFERENCES BARKER, J. (1936). Proc. Roy. Soc. B, 119, 453. DOVE, W. F., MURPHY, E. F. & AKELEY, R. V. (1943). Genetics, 28, 72. EssELEN, W. B., LYONS, M. E. & FELLERS, C. R. (1942). Bull. Mass. Agric. Exp. Sta. no. 390. LAMPITT, L. H., BAKER, L. C. & PARKINSON, T. L. (1945). J. Soc. Chem. Ltd., Lond., 64, 18. MAPSON, L. W. (1948). Unpublished. MAPSON, L. W., CRUICKSHANK, E. M. & CHEN, Y. (1949). Biochem. J. 45, 171. OLLIVEK, M. (1938). Analyst, 63, 2. PETT, L. B. (1936). Biochem. J. 30, 1228. RAY, S. N. (1934). Biochem. J. 28, 996. SCHEUNERT, A., RESCHKE, J. & KOHLEMANN, E. (1937). Bioclieni. Z. 29O, 313. SMITH, A. M. & GILLIES, J. (1940). Biochem. J. 34, 1312.