the organic acids of rhubarb (rheum …preliminary information with respect to the oxalic, gmalic,...

THE ORGANIC ACIDS OF RHUBARB (RHEUM HYBRIDUM)*

II. THE ORGANIC ACID COMPOSITION OF THE LEAVES

BY GEORGE W. PUCHER, HAROLD E. CLARK,? AND

HUBERT BRADFORD VICKERY

(From the Biochemical Laboratory of the Connecticut Agricultural Experiment Station, New Haven)

(Received for publication, November 24, 1936)

Although it has been known since the time of Scheele that higher plants contain considerable amounts of a variety of organic acids, surprisingly little definite information is available regarding the origin, function, or fate of these substances in the organism. Bennet-Clark (1) concludes his recent exhaustive review of the role of organic acids in plant metabolism with the statement:

“General pronouncements on the significance of the plant acids are at present probably of little value. . . The true significance of the acid metabolism of plants may perhaps lie close to Kostytchev’s later point of view that these acids are the building stones from which some of the complex plant products originate. . . The plant acids are possibly convertible into amino acids and proteins, complex products such as the alkaloids, and by reduction into the parent substances of the fats and also into carbohy- drates. Thus they may form some essential links in the processes by which the energy of respiration is transferred to the varied synthetic activities of the plant. It is certainly clear that they are by no means waste products of metabolism.”

The present investigation was undertaken in order to obtain preliminary information with respect to the oxalic, Gmalic, and citric acid content of rhubarb leaves. We were interested in a number of phases of the subject. The matter of the alleged presence of dl-malic acid in this species has been dealt with in the

* The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Wash- ington.

t National Research Council Fellow, 1933-35.

605

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606 Organic Acids of Rhubarb. II

preceding paper (2). In this paper we propose to discuss the quantities of the several organic acids in the petiole, main vein, and leaf blade tissues, the concentration gradients of the acids in the parts of the leaf, the relation between the concentration of the total acids in the tissues and the titratable acidity, and also the relationship between the concentration of t,he several acids and of the ammonia.

The methods of analysis employed (3) have been developed in t,his laboratory specifically for the purpose of conducting surveys of the organic acid composition of green plants. The determinations are made upon a solution obtained by ether extraction of a sample of the previously dried tissue moistened with sufficient dilute sulfuric acid to bring it to pH 1. Under these conditions the organic acids present are quantitatively extracted. The acids are transferred to dilute aqueous alkali, the ether is evaporated, and the solution is made to a def?nite volume. The individual determinations are made on aliquot parts of this solution. A detailed description of the samples of rhubarb studied is given in the preceding paper.

Results

Table I shows the quantities of each constituent in gm. or milli- equivalents per single leaf or leaf part, or, in t.he case of Sample I, per single bud. Table I, accordingly, gives a survey of the data in terms of a biological unit and permits inferences to be drawn with respect to the actual changes in quantity with age, as well as comparisons of the conditions in leaves of similar age developed from rhizomes of increasing age.

The data in Sections 1 and 2 show that the leaves of Sample IV were slightly heavier than those of Sample III, but this difference was due to greater hydration; the dry weight was actually less save in the petiole. It is clear that the essential change in- volves an increase in both dry weight and water in the petiole during the 14 day interval between collections. The leaves of Sample VI, collected 45 days later, had developed from buds during the 35 days previous to collection. They were materially heavier than the previous sample, and contained more dry matter in eachpart, and considerably more water. The last sample taken 41 days later, and which had also developed from buds in the interval,


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Pucher, Clark, and Vickery

TABLE I

Organic Acids oj Rhubarb Leaves

The data are expressed as gm. or milli-equivalents per individual leaf or leaf part.

Sample No .................................. Collection date .............................. ‘I interval, days ....................

Section 1. Fresh weight, gm. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 2. Dry weight, gm., ...... Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 3. Water, gm. Blade. ....... .................. Veins ........................... Petiole ......................... Whole leaf ......................

Section 4. Total organic acids, m.-eq.

Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 5. Oxalic acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 6. Citric acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 7. Malic acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Wholeleaf ......................

*pi. 9

3.11

0.488

2.62

0.97

0.035

0.26

0.05

III

‘“z’i 3o

IV ““3’5 I4

VI

‘“io” 28

VII 43 8

24.1 10.3 30.4 31.1 9.6 9.7 13.1 10.4

40.9 48.8 73.9 59.9 74.6 78.8 17.4 31.4

3.48 2.46 3.53 4.37 0.86( 0.69: 0.96( 0.995 2.53 2.66 4.15 4.66 6.87 5.82 8.64 10.0

20.6 17.9 26.9 26.7 8.70 9.05 12.1 9.4

38.4 16.2 69.7 55.3 67.7 73.1 08.7 91.4

7.11 5.89 10.1 13.4 2.72 2.79 4.29 4.11

11.6 14.8 23.3 22.5 21.4 23.5 37.7 40.0

1.45 1.74 5.17 6.50 0.66 0.79 1.53 1.36 3.07 3.52 6.30 6.60 5.18 6.05 13.0 14.5

1.32 0.76 0.85 1.22 0.23 0.17 0.26 0.41 0.55 0.56 1.14 2.30 2.10 1.49 2.25 3.93

0.85 0.97 1.41 2.29 1.08 1.54 2.06 1.92 6.43 10.0 15.4 12.6 8.36 12.5 18.9 16.8


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TABLE I-Concluded

Sample No.. Collection date.. “ interval. days. _.

Section 8. Total known acids, m.-eq.

Blade ........................... Veins ........................... Petiole .......................... Whole leaf .......................

Section 9. Unknown acids, m.-eq. Blade ........................... Veins ............................ Petiole .......................... Whole leaf .......................

Section 10. Ash, gm. Blade ........................... Veins ............................ Petiole .......................... Whole leaf .......................

Section 11. Ratio of total organic acids to ash

Blade ........................... Veins ............................ Petiole .......................... Whole leaf ......................

0.34

0.63

-

III ipr. 30

21

VII hi *

3.62 3.47 7.43 10.0 1.97 2.50 3.85 3.69

10.0 14.0 22.8 21.5 15.6 20.0 34.1 35.2

3.49 2.42 2.67 3.44 0.75 0.29 0.44 0.42 1.60 0.80 0.50 1.00 5.84 3.51 3.61 4.86

0.25( 0.191 0.33! 0.0% 0.07! 0.12: 0.23fv 0.261 0.5Of 0.57! 0.54 0.96t

0.396 0.110 0.487 0.993

27.8 30.4 29.6 31.3 35.3 34.9 29.2 55.2 46.0 37.0 43.4 38.9

33.9 37.4 46.3 40.4

showed evidence of dehydration coupled with increase in dry matter in each part. In general, therefore, young rhubarb leaves appear to pass through a stage of high hydration. Leaves that develop at a later part of the season are relatively high in dry matter and low in water content.

Section 4 of Table I shows that the total organic acidity of the leaves increases with age, and also that late developing leaves contain a larger amount of acids than earlier leaves. An inter- esting relationship exists between the quantities of organic acids and of ash, not only with respect to each leaf part, but also with respect to the leaf as a whole. The ratios are given in Section 11 of Table I and show that the variations in the amounts of acid are closely similar to the variations in the amounts of ash. The ratios for each leaf part are substantially constant, though the ratios are different for the different parts.


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Pucher, Clark, and Vickery 609

The distribution of the total acidity in the several parts of the leaf is to only a minor extent affected by age. Thus 30f4 per cent of the acidity is found in the blade, llf4 per cent in the veins, and 59%4 per cent in the petioles, when calculations for the en- tire set of data are made. Such a constancy of distribution in the leaf, regardless of age or period of development, suggests that a mechanism exists which controls this distribution. In view of the relationship between acidity and inorganic ash already mentioned, it seems probable that this mechanism operates in the same manner on both acids and inorganic cations. Whether organic cations likewise share in the relation is not known.

The data for oxalic acid (Section 5) show an increase with age in each part of the leaf, but the most striking feature is the much higher oxalic acid content of the leaf blades that were developed in the later part of the season. These contained nearly 5 times as much oxalic acid as the youngest leaf blades, while the oldest petioles contained about twice as much as the youngest. The blades of the older leaves contained approximately the same amounts of oxalic acid as the petioles of the same leaves, but more oxalic acid was present in the petioles than in the blades of the younger leaves. This observation is apparently contrary to the findings of Angerhausen (4) who states that the oxalic acid of the leaf blade is greater than that of the petiole. The discrepancy is more apparent than real. Angerhausen based his conclusion on data expressed on a concentration basis; i.e., per cent of the fresh weight. As will appear below, our own data, when expressed in an analogous manner, give rise to a similar but misleading conclusion.

The rapid synthesis of oxalic acid in the first 21 days of development of the leaf indicates that reactions in which oxalic acid is an end-product form an important phase of the acid metabolism of very young tissue. A similar observation has been made on tobacco plant tissue (5).

The situation with respect to citric acid is somewhat different. The blades of Sample IV contained considerably less citric acid than those of Sample III, and the increase in the blades of the older leaves is of a minor nature. The main veins contained approximately the same absolute amount of citric acid, regardless of the age of the plant. The petioles of the later developed leaves,


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TABLE II Organic Acids of Rhubarb Leaves

The data are expressed as gm. or milli-equivalents per 100 gm. of water in the whole leaf or leaf part.

Sample No ................................... Collection date ............................... “ interval, days .....................

Section 1. Dry weight, gm. Blade ........................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 2. Total organic acids, m.-eq.


Section 3. Oxalic acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 4. Citric acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 5. Malic acid, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

Section 6. Total known acids, m.-eq.


Section 7. Unknown acids, m.-eq. Blade .......................... Veins ........................... Petiole ......................... Wholeleaf ......................

Section 8. Ash, gm ................ Blade .......................... Veins ........................... Petiole ......................... Whole leaf ......................

-

-

. *

-

18.7

37.0

1.34

9.9

1.9

13.14

23.86 i

- III

‘“z’i 3o

.6.9 14.2 13.1 16.4 7.5 7.7 8.0 LO.6 6.6 5.8 5.95 8.4 .0.15 7.96 7.95 10.97

14.6 33.0 37.4 48.5 11.2 30.8 35.6 43.8 %0.2 31.9 33.4 40.7 51.64 32.1 34.6 23.9

7.05 9.7 19.2 24.4 7.6 8.7 12.7 14.5 7.9 7.6 9.1 11.9 7.65 8.28 11.95 16.0

6.4 4.4 3.2 4.6 2.6 1.9 2.2 4.4 1.3 1.2 1.6 4.2 3.10 2.04 2.07 4.30

4.1 5.45 5.25 8.6 12.4 17.0 17.1 20.5 16.7 21.6 22.2 22.8 12.3 17.1 17.4 18.4

17.55 19.55 27.65 37.6 22.6 27.6 32.0 39.4 25.9 30.4 32.9 38.9 23.05 27.4 31.4 38.7

17.05 13.45 9.75 10.9 8.6 3.2 3.6 4.4 4.3 1.5 0.5 1.8 8.59 4.7 3.2 5.2

1.25 1.03 1.26 1.49 1.00 0.87 1.02 1.17 0.62 0.56 0.73 0.88 0.85 0.74 0.89 1.09

VI rune 28

80

VII 4wi 8

610


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however, contained about 4 times as much citric acid as those of the youngest.

Malic acid, like oxalic acid, occurs in larger amounts in all parts of the leaves of later development than in the younger leaves, approximately twice as much being present in each leaf part of Sample VII as in Sample III.

The unknown acids, that is the difference between the total organic acidity and the sum of the oxalic, citric, and malic acids, occur in greater amount in the young tissues than in the older, and a larger amount occurs in the blades than in the veins or petioles.

In general, the predominating acid of the leaf blade is oxalic; the group of unknown acids taken together makes up a quantity even greater than the oxalic in the youngest leaves, but is distinctly less important quantitatively in the leaves of later development . Malic and citric acids are present in smaller amounts.

The predominating acid of the vein tissue is malic acid with oxalic acid in second place. Citric acid and the group of unknown acids occupy minor positions.

The petiole tissue likewise shows a marked preponderance of malic acid, oxalic acid again being a close second in quantity. Citric acid is in third place, and, save in the youngest tissue, the unknown group is present only in small amounts.

The acid composition of each part of the leaf undergoes con- tinuous change as the age of the root system increases, and, in view of the constancy of the relative amounts of total organic acids in the several parts of the leaf, it is clear that there is a close interrelationship in the metabolism of the three chief acids.

The data in Table II have been calculated in terms of the concentration in gm. or milli-equivalents per 100 gm. of water in each leaf part, or in the whole leaf. The figures are therefore analogous to and are also closely similar in order of magnitude to results calculated on the basis of percentage of the fresh weight. They refer more accurately, however, to the fact that the organic acids occur for the most part in solution in the cells as acid salts’

1 If it be assumed that the acids dissociate in the cell sap essentially as they do in water, then at pH 3.3, the approximate reaction of petiole sap, malic acid is present to the extent of 62 per cent as free acid, 35 per cent as acid salt, and 2 per cent as neutral salt; oxalic acid, 2 per cent as free acid, 96 per cent as acid salt, and 8 per cent as neutral salt; citric acid,


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and permit comparisons of the relative concentrations of the acids in the different parts of the leaf.

Section 2 of Table II shows that organic acids occur in relatively high concentration in the bud tissue, at a somewhat lower concentration in the young leaves, but at gradually increasing concentration in the leaves of later development until ultimately the initial concentration in the bud is exceeded. Bennet-Clark and Woodruff (6) likewise observed a high concentration of acids in young tissue and a decrease with age. Although there is a slight concentration gradient diminishing from blade to vein to petiole in all the samples, the most striking feature of the data as a whole is the approximate constancy of the concentration of total acids in all parts of the leaf regardless of the age of the root system at which the leaves developed. Only the last sample shows a distinctly higher concentration than the others.

The data for the individual acids can be most clearly summa- rized in terms of the obvious concentration gradients present in the leaf structure. The concentration of oxalic acid, although essentially constant in all parts of the leaves of Sample III, shows a marked increase from petiole to vein to leaf blade in all the other samples. In the leaves of late development the concentration in the blade is more than twice as great as in the petiole. Citric acid shows a gradient in the same direction in the young leaves, but this becomes less apparent in the leaves of intermediate age, and disappears in the oldest leaves. In all cases, however, the concentration of citric acid is low compared to that of oxalic acid.

The unknown organic acids are also distributed in accordance with a concentration gradient which increases from petiole to blade, and in this case the gradient is exceedingly steep, there being a very much higher concentration of these acids in the

37 per cent as free acid, 53 per cent as diacid salt, 8 per cent as monoacid salt, and 1 per cent as neutral salt. At pH 4.3, the approximate reaction of the blade tissue, malic acid is present to the extent of 13 per cent as free acid, 73 per cent as acid salt, and 13 per cent as neutral salt; oxalic acid, 60 per cent as acid salt, and 40 per cent as neutral salt; citric acid, 5 per cent as.free acid, 42 per cent as diacid salt, 48 per cent as monoacid salt, and 6 per cent as neutral salt. These calculations disregard the possibility that cations may be present which precipitate a portion of one or more of the acids.


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blade than in the petiole. What relationship these concentration gradients bear to possible movements of the acids within the leaf structure cannot be stated from the present evidence. Experiments such as those of Maskell and Mason (7) with the cotton plant would be required to shed light on this point.

I-Malic acid is distributed in the leaves of the rhubarb in a manner quite the opposite to that of the other acids. In all cases there is a higher concentration in the petiole than in the blade with the veins occupying an intermediate position in this respect, but nearer to the petiole than to the blade. The concentration in the blade is, in fact, of the same order as that of the citric acid, whereas the concentration in the petiole is in all cases very high, far exceeding that of the oxalic acid. It is because of the existence of this steep opposing gradient that the total acid concentration in all parts of the leaf is not far from constant.

A conclusion very different from this is reached if reliance is placed on the titratable acidity as a measure of the total acids present. Steinmann (8) and also Culpepper and Caldwell (9) have presented such data and arrive at the conclusion that the acid concentration in the petiole is materially higher than that in the leaf blade. The difference can be readily explained, however. Although the total organic acidities of the blade and petiole are not far apart, the hydrogen ion activity of the petiole is much greater than that of the blade (see Table IV) owing to the presence of a higher concentration of inorganic cations in the blade tissue (see Table II, Section 8). Moreover, malic acid occurs in high concentration in the petiole, and the reaction is such that about 60 per cent of it is present as free undissociated acid. Accordingly, direct titration of extracts of the two tissues might be expected to show an appreciable difference.

The data in Table II can be shown to illustrate this. If it be assumed that the unknown organic acids are dibasic and of a strength similar to that of malic acid, and further, that the titration end-point is at pH 8, a figure which approximates to the titratable acidity can be calculated. The data in Table III were obtained in this way, and it is clear that in all cases the calculated titratable acidity of the petiole is materially greater than that of the leaf blade, a result in complete agreement with the findings of the authors mentioned above.


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Relationship between Organic Acids and Ammonia-The detoxi- cation of ammonia in plants by synthesis of an amide (asparagine or glutamine) is a well known phenomenon. It has been studied by Prianischnikow (10) and by Mothes (11) in many species, and has been observed in this laboratory in the beet (12), the tobacco (13), and the tomato plant (14). An alternative method of de- toxication has been suggested by Ruhland and Wetzel, who main- tam that plants possessed of highly acid saps detoxify ammonia by simple neutralization with an organic acid, both oxalic and malic acids being so used. Ruhland and Wetzel (15) classify rhubarb as a typical acid plant, and state that a rapid deamina- tion of amino acids occurs during the development of the leaf ac- companied by the production of I-malic acid and of ammonia in

TABLE III

Total Organic Acidity and Calculated Titmtable Acidity of Rhubarb Leaves

The data are expressed in milli-equivalents per 100 gm. of water in each leaf part.

Sample No.

III 34.6 10.9 IV 33.0 14.0 VI 37.4 15.0 VII 48.5 20.8

Blade P&i&

Total organic Titratable acidity acidity

Total organic Tit&able acidity acidity

30.2 21.2 31.9 23.2 33.4 23.2 40.7 27.8

approximately equivalent quantities. Under certain conditions of culture, they found that the ammonium nitrogen may increase to 50 per cent or more of the total nitrogen of the tissue with a correspondingly high production of malic and oxalic acids.

These conclusions have been seriously called in question by Bennet-Clark and Woodruff (6) on several grounds. They point out that Ruhland and Wetzel have not described the methods by which their results were obtained, and show that some of the published data are in conflict with the laws of physical chemistry. They were also able to show that certain of Ruhland and Wetzel’s claims are based upon a confusion of the concentration of the organic acids in the tissues with the actual quantity present in an individual leaf or plant. Bennet-Clark and Woodruff themselves


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found that the ammonium content of the rhubarb plants they studied bore no relationship to the organic acid present.

The data in Table IV show the concentration of ammonia and of the individual organic acids in the samples of rhubarb tissue we have examined. Although an increased ammonia concentration is clearly evident in the leaves of late development, there is no quantitative relationship between the ammonia and any one of the three determined organic acids, with the exception of the citric acid in the petioles. The possibility that citric acid behaves as a detoxicating substance for ammonia in the sense of Ruhland

TABLE IV

Concentration oj Organic Acids and of Ammonia in Rhubarb Tissue

The data are expressed in milli-equivalents Pel r 100 gm. of water.

Buds Blade

Petiole

Rhizome

SaNmoqle

I III IV VI VII III IV VI VII VIII VIII

1

-

Reaction PH

NHs-N Total

organic acids

Oxalic acid

Citric acid

5.22 0.67 37.0 1.34 1.9 9.9 4.73 0.71 34.6 7.05 4.1 6.4 4.27 0.91 33.0 9.7 5.5 4.4 4.21 2.39 37.4 19.2 5.3 3.2 4.10 3.06 48.5 24.4 8.6 4.6 3.26 0.62 30.2 7.9 16.7 1.3 3.20 1.23 31.9 7.6 21.6 1.2 3.26 1.82 33.4 9.1 22.2 1.6 3.33 1.64 40.7 11.9 22.8 4.2 3.67 4.02 36.0 9.6 19.1 4.0 5.56 2.26 82.5 45.7 2.9 4.6

and Wetzel is rendered remote, however, by the data for the leaf blades which show a decrease in citric acid concentration with increasing age of the plant coupled with an increase in ammonia concentration. Certainly none of our specimens contained any such quantities of ammonia as would correspond to the 50 per cent of the total nitrogen mentioned by Ruhland and Wetzel, and there is no correspondence whatever between the ammonia content of the samples of rhubarb we have studied and those described by them. Our experience in this connection confirms and extends that of Bennet-Clark and Woodruff.


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SUMMARY

The rhubarb leaf contains kmalic, oxalic, and citric acids together with acids of unknown nature. The composition differs in different parts of the leaf and is profoundly influenced by the age of the leaf and by the season in which it has developed.

The group of unknown acids predominates in the blades of the younger leaves, oxalic acid being present in next smaller amount. In the blades of leaves developed late in the season, oxalic acid predominates over the unknown acids. I-Malic and citric acids are present in small amounts in the blades.

The predominating acid of the main veins is Z-malic with oxalic in second place. Citric acid and the unknown acids occupy minor positions.

The predominating acid of the petiole is Lmalic with oxalic in second place. Citric and the unknown acids are present in small amounts.

The concentration data show that oxalic, citric, and the unknown acids occur in a concentration gradient that increases from petiole to vein to blade. I-Malic acid, on the other hand, is present in a concentration gradient that decreases from petiole to vein to blade. As a result, the concentration of the total organic acids is not far from constant in all parts of the leaf; there is only a slight gradient of the total organic acidity which increases from petiole to blade. It is shown that this conclusion is not at variance with the results of direct titration of extracts of the tissues; the titratable acidities of the blades and petioles calculated from the present data are in agreement in relative order of magnitude with those reported by Steinmann and by Culpepper and Caldwell.

Determinations of the ammonia in the t’issues showed that the concentration is in all cases quite low, and that there is no correla- tion whatever with the concentration of any individual acid nor with the total acidity. This observation confirms and extends the results of Bennet-Clark and Woodruff and, like theirs, does not agree with the conclusions of Ruhland and Wetzel.

BIBLIOGRAPHY

1. Bennet-Clark, T. A., New Phytologist, 33,37, 128, 197 (1933). 2. Pucher, G. W., Clark, H. E., and Vickery, H. B., J. Biol. Chem., 117,

699 (1937).


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3. Pucher, G. W., Vickery, H. B., and Wakeman, A. J., Ind. and Eng. Chem., AnaE. Ed., 6,140, 288 (1934). Pucher, G. W., Vickery, H. B., and Leavenworth, C. S., Ind. and Eng. Chem., Anal. Ed., 6,190 (1934); 7, 152 (1935).

4. Angerhausen, J., Z. Untersuch. Nahrungs- u. Genussmittel, 39, 81, 122 (1920).

5. Vickery, H. B., Pucher, G. W., Leavenworth, C. S., and Wakeman, A. J., Connecticut Agric. Exp. Rat., Bull. $74 (1935).

6. Bennet-Clark, T. A., and Woodruff, W. M., New Phytologist, 34, 77 (1935).

7. Maskell, E. J., and Mason, T. G., Ann. Bot., 43, 205,615 (1929); 44, 1, 233,657 (1930).

8. Steinmann, A. B., 2. Bot., 9,1 (1917). 9. Culpepper, C. W., and Caldwell, J. S., Plant Physiol., 7, 447 (1932).

10. Prianischnikow, D., Ber. deutsch. bot. Ges., 40, 242 (1922); Biochem. Z., 160,407 (1924).

11. Mothes, K., 2. wissensch. Biol., Abt. E, Planta, 1,472 (1926). 12. Vickery, H. B., Pucher, G. W., and Clark, H. E., Plant Physiol., 11,

413 (1936). 13. Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth,

C. S., Carnegie Inst. Washington, Pub. No. 445 (1933). 14. Vickery, H. B., Pucher, G. W., and Clark, H. E., Science, 80,459 (1934). 15. Ruhland, W., and Wetzel, K., 2. wissensch. Biol., Abt. E, Planta, 3,

765 (1927); 7,503 (1929). by guest on April 13, 2020

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Bradford VickeryGeorge W. Pucher, Harold E. Clark and Hubert

THE LEAVESORGANIC ACID COMPOSITION OF

(RHEUM HYBRIDUM): II. THE THE ORGANIC ACIDS OF RHUBARB

1937, 117:605-617.J. Biol. Chem.

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