THE EFFECT OF CARBON DIOXIDE UPON THE pH AND CERTAIN NITROGEN FRACTIONS OF THE

SUGAR-BEET PLANT

BY J. M. FIFE AND V. L. FRAMPTON

(From the United States Department of Agriculture, Bureau of Plant Industry, Division of Sugar Plant Investigations, Riverside, California)

(Received for publication, February 4, 1935)

It is the general belief that the hydrogen ion activity of the cell sap is increased by the accumulation of carbon dioxide. For example, Willaman and Beaumont (5) observed that when carbon dioxide was allowed to accumulate in an atmosphere in which twigs, tubers, or grain was stored, the rate of carbon dioxide production by these tissues decreased in a logarithmic ratio. In the case of the twigs, the amount of carbon dioxide produced was proportional to the logarithm of time. The rate of production of carbon dioxide immediately assumed a far higher value when the accumulated carbon dioxide was removed. In seeking an explana- tion, these investigators ((5) p. 52) state: “Another possible explanation was suggested by Dr. R. A. Gortner. It is that the accumulation of CO2 in the tissues increases the hydrogen-ion concentration in the latter; that this brings the proteins of the protoplasm nearer to their isoelectric point, and hence increases its permeability, which is responsible (perhaps through increased enzyme activity) for an actual increased rate of CO, production.” A further quotation from Willaman and Beaumont ((5) p. 53), “That the acidity of the tissue fluids is increased by the accumu- lation of CO2 is well known, and does not need a specific illustra- tion,” shows how generally this belief is held.

This idea is apparently supported by the fact that carbon dioxide increases the hydrogen ion activity of the expressed plant juice. The fact that the hydrogen ion activity of the cell sap within t.he living plant is not increased when the plants are exposed to high concentrations of carbon dioxide will be shown in this paper and an explanation of the reactions involved will be suggested.

643

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644 Nitrogen of Sugar-Beet

Magness and Diehl (1) observed that in some cases the acidity of fruit was decreased on treatment with carbon dioxide. They also observed that the titratable acidity of the winesap apple, expressed as cc. of 0.1 N acid per 10 gm. net weight of tissue, decreased from 6.55 to 6.14 with a treatment of 100 per cent car- bon dioxide.

Thornton (4) reported that the treatment of various types of plant tissues with carbon dioxide resulted in a decrease in the hydrogen ion concentration of the juice extracted from those tissues.

The studies on the effect of carbon dioxide reported in this paper were conducted in 1931 and 1932 in connection with investi- gations on the chemical nature of resistance in sugar-beets to the curly top disease. In these investigations pH measurements of the juice of various strains of sugar-beets and other plants affected by the disease were made. The first pH determinations were made with the quinhydrone electrode. Juice from the leaves of the various strains of sugar-beets (mass selections), and from individual plants of the same strains, varied in pH from 6.07 to 6.88. With the same electrode, leaf juice from Chenopodium murule (extremely resistant to curly top) consistently gave pH values varying from 7.3 to 8.01 immediately after extraction. In view of this apparent difference in pH and the greater disease resistance of the Chenopodium mu&e, it seemed probable that if the pH value of this plant could be temporarily reduced it might be rendered susceptible to the disease.

In view of the acid nature of carbon dioxide gas, an attempt was made to reduce the pH of Chenopodium murale by subjecting the entire plant to an atmosphere rich in this gas. When the plant was removed from the gas chamber and the juice expressed imme- diately, it was observed that the pH had not decreased as was

1 Further pH determinations made at a later date revealed that these values, although constant, were high and misleading. If the juice ex-

tracted from Chenopodium murale were allowed to stand in the refrigerator for 3 days before pH determinations were made, true values were obtained. It is quite probable that certain soluble proteins are responsible for the

abnormal results. It was observed that during storage a precipitate formed. True pH values were obtained (when precipitation was complete) even in the presence of the precipitate. Storage of thd beet juice in the

refrigerator had no effect on the pH value.

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J. M. Fife and V. L. Frampton 645

expected, but had greatly increased. This experiment was re- peated several times with the same result. Further investigations revealed that sugar-beet plants responded in the same manner, the pH being increased as much as 2 units in some cases.

This unexpected decrease in the hydrogen ion concentration of the juice of these plants, when exposed to a high concentration of carbon dioxide, encouraged more detailed investigations into the nature of the chemical reactions involved because of a possible bearing on resistance to curly top.

Methods

The plants receiving the carbon dioxide treatment were placed under bell jars and definite amounts of carbon dioxide were applied for different lengths of time at room temperature. The experi- ments were carried out both in the dark and in the light. Juice was extracted from the controls and from the carbon dioxide- treated plants with the aid of a hydraulic press immediately upon their removal from the bell jars.

The pH measurements were made with the glass electrode, the hydrogen electrode, and the quinhydrone electrode. The glass electrode used was similar to that described by Robertson (3).

Results

In the first experiments, the entire plants were subjected to an atmosphere containing 50 per cent carbon dioxide for 24 hours. The plants were then removed and the juice expressed imme- diately. Table I shows the effect of a high concentration of carbon dioxide on the pH of the plant. These data are typical of more than 100 experiments which were conducted over a period of 2 years. In every case where the concentration of carbon dioxide was 10 per cent or greater, the pH of the juice, extracted from the plants immediately after treatment, was found to be higher than that of the juice from the untreated plants. In some of these experiments, the hydrogen ion activity of the juice of beet leaves was decreased as much as loo-fold by treating the plant with a high concentration of carbon dioxide. The results were of the same order whether the carbon dioxide exposure was made in the light or in the dark. When the entire plant was treated, juice extracted from the leaves, petioles, and roots showed an increase

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646 Nitrogen of Sugar-Beet

in pH, as shown in Table I which gives the results of representative tests.

The pH of juice extracted from carbon dioxide-treated plants does not change on standing, yet it responds to carbon dioxide treatment in the normal way. It was found, for example, that when the expressed juice from carbon dioxide-treated beet leaves having pH of 7.45 was saturated with carbon dioxide, the pH decreased to 5.93. When the dissolved carbon dioxide was removed, the pH returned to its original value of 7.45. Juice expressed from normal beet leaves responded in a similar manner, going from pH 6.55 to 5.75 when saturated with carbon dioxide, and returning to its original pH of 6.55 when the gas was removed.

TABLE I

pH of Juice Expressed from C&Treated and Normal Plants, As Determined

by Quinhydrone Electrode

Plant

Chenopodium murale Sugar-beet

Leaves Blades Petioles Roots

pH of expressed juice

Normal CO&mated

7.35* 8.11 6.26 7.36 5.80 7.05 6.11 6.57

* See foot-note 1.

Neither macerated nor plasmolyzed tissues responded to carbon di- oxide treatment as did the living plant; i.e., by an increase in pH.

The fact that high concentrations of carbon dioxide will effect an appreciable change in the pH of other plants was demonstrated with several species of plants susceptible to curly top. In these tests the pH determinations were made with the glass, the hydro- gen, and the quinhydrone electrodes, as shown in Table II.

All plants that have been tested thus far have responded in the same manner to carbon dioxide treatment. The degree to which the pH increases on carbon dioxide treatment varies widely and appears to be independent of the pH of the normal tissue. The chemical reaction responsible for this increase in pH is very effective in view of the buffering of the normal cell sap. To illustrate, the pH of Oxalis martiana was found to be 2.17 nor-

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J. M. Fife and V. L. Frampton 647

mally. This value was increased to 2.74 by treating the plant with carbon dioxide, despite the high buffer index (0.070) of the juice.

The pH values obtained with the glass electrode are probably more reliable than those with the quinhydrone or hydrogen electrodes, because the glass electrode is unaffected by oxidizing agents, catalysts, or poisons. From Table II it is evident that the quinhydrone electrode is suitable for pH measurements on the

TABLE II

pH of Expressed Juice of CO1-Treated and Normal Plants

Plant Glass

Norma:

Asparagus oficinalis .............. 6.09 Atriplex bracteosa. ............... 6.17 Beta vulgaris ...................... 5.95 Chenopodium murale. ............. 6.39’ Erodium cicutazium .............. 5.58 Lycopersicon esculentum .......... Matthila incana .................. 5.20 Nicotiana tabacum ................ 5.25 Oxalis martiana ................... 2.24 Solanum nigrum. ................. 5.60 Stellaria media ................... 6.10

COz- treatec

6.51 6.43 6.87

6.65 5.75

5.46 5.52 2.63

6.10 6.77

Electrode

Hydrogen -i- Quinhydrone

6.14 6.72 6.07 7.20

6.45* 6.89 5.35 5.91

5.12 5.36 5.22 5.45

1.98 2.46 5.59 6.13 6.09 7.32

6.47 6.80 6.08 6.91

6.36# 6.71 5.54 6.03 5.12 5.34

5.23 5.46 5.36 5.66 2.17 2.74 5.36 6.05 6.09 6.84

car treakd

* It is of interest to point out the close agreement in pH of Chenopodium murale as determined by the different electrodes. In this instance the

juice was extracted and allowed to remain in the refrigerator for 3 days before the pH determinations were made with the quinhydrone electrode.

juice of the beet leaf and of other plants. In view of this fact, the subsequent measurements of the changes in pH on the juice of the beet leaf were made with the quinhydrone electrode.

Such striking changes in the hydrogen ion concentration of the plant juice, due to carbon dioxide treatment, must be the result of certain chemical reactions catalyzed by the plant. It appears logical that, if carbon dioxide or the increase in hydrogen ion concentration in the cell sap due to the dissolved carbon dioxide is the catalyst for these reactions, the rate of response of the plant

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Nitrogen of Sugar-Beet

and the extent to which the reaction takes place will depend on the concentration of carbon dioxide dissolved in the cell sap and the duration of treatment.

In view of the fact that the diffusion of carbon dioxide into the leaves takes place through the intercellular spaces, the rate of diffusion of the gas and the amount dissolving in the cell sap depend on the partial pressure of carbon dioxide applied. A determination of the speed with which the hydrogen ion activity is altered in the plant (presumably counteracting the acidic effect of the carbonic acid formed in the cell sap) is of considerable importance in that it may throw light on the rate at which certain reactions proceed in plants.

In order to determine the rate at which beet plants will respond to carbon dioxide treatment, a large number of beet leaves were collected and divided into six lots, one of which was the control. The leaves were placed under bell jars and subjected to an atmos- phere containing 40 per cent carbon dioxide. At the end of each of the following periods-30, 60, 90, 120, and 180 minutes-one lot of leaves was removed, the midribs and petioles discarded, the juice extracted immediately, and the pH determined.

Then, to determine the rate at which the beet plant will return to normal after carbon dioxide treatment, a large sample of beet leaves was placed under a large bell jar and exposed to an atmos- phere containing 40 per cent carbon dioxide for 1 hour, after which it was removed. A small number of leaves were extracted imme- diately, while the remainder was allowed to stand in the laboratory. At 15, 30, 60, 90, and 180 minute intervals after their removal from the bell jar, the juice was extracted from a small number of leaves and the pH determined.

Curve A of Fig. 1 shows the rate at which the beet plant re- sponded to carbon dioxide treatment, while Curve B shows the rate at which the pH of the plant returned to normal when the carbon dioxide treatment was discontinued. It is evident that the beet leaf catalyzes the reaction responsible for the pH change at a very rapid rate in either direction.

In view of the fact that only the partial pressure of carbon dioxide inside the cells would be effective in causing the plant to react in such a striking manner, a study of the effect of different concentrations of carbon dioxide on the reaction of the plant was made to determine how far the reactions would proceed.

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J. M. Fife and V. L. Frampton 649

A large number of beet leaves were collected and divided into seven lots, one of which served as the control. Each of the remaining lots of leaves was subjected to a definite concentration of carbon dioxide for 30 minutes. The samples of leaves were then removed from the bell jars, the juice extracted immediately, and the pH determined. The experiment was repeated, except that in this case each lot of leaves was allowed to remain in its

Z/O

6.90

x Q

6.70

650 NORMAL PLANT- *

T/ME /N U INU TES

0 30 60 90 /20 /SO I80

Fra. 1. The rate at which sugar-beet plants respond to and recover from carbon dioxide treatment. Curve A, the rate of response to carbon dioxide treatment; Curve B, the rate at which carbon dioxide-treated plants returned to normal.

respective concentration of carbon dioxide for a period of 1 hour. The results of these experiments are shown in Fig. 2.

It is apparent that the partial pressure of carbon dioxide is not the limiting factor in determining the extent to which the pH will change, for 20 per cent is almost as effective as 40 or 60 per cent in producing a change. At 80 per cent carbon dioxide (Fig. 2) the reaction is greatly intensified in both the 30 and 60 minute periods of treatment. This is very striking, especially when compared to the change in pH which occurs in 100 per cent carbon

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650 Nitrogen of Sugar-Beet

dioxide. It is important to note that at 100 per cent carbon dioxide an increase in pH of the extracted juice was obtained only when the plants were exposed to the gas for a short period (approx- imately 1 hour). With an exposure of 4 or 5 hours the pH of the extracted juice was found to be considerably decreased below that of untreated controls.

The evidence tends to show that the changes which take place in the beet leaf during treatment with high concentrations of carbon dioxide are not due to translocation. Leaves or petioles

PER CENT CARBON D/OX/DE 10 20 30 40 50 60 70 80 90

Fro. 2. The pH change in relation to the concentration of carbon dioxide applied to the beet plant.

which are removed from the remainder of the plant respond as readily and to the same degree to carbon dioxide treatment as leaves and petioles attached to the plant. The reactions, then, are local in character. However, this reaction is catalyzed by all parts of the plant. It is also evident that the reaction is not dependent on radiant energy, because the change takes place as readily in the dark as in the light.

In casting about for an explanation of this phenomenon and the possible reactions involved in the process, certain nutrition experi- ments in which beet plants received different amounts of nitrogen

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J. M. Fife and V. L. Frampton 651

furnished one clue to the type of compounds involved. It was observed that leaves taken from a series in which the plants received an abundance of nitrogen responded to a greater degree (as measured by the increase in pH on carbon dioxide treatment) than leaves removed from plants which were nitrogen-starved.

In view of these facts, it was suspected that perhaps the nitrog- enous compounds in the plant were involved in the reactions which cause a decrease in the hydrogen ion activity of the plant. Experiments were carried out to determine what role the nitrog- enous compounds play in these reactions.

A large number of beet leaves were collected, part of which was treated with carbon dioxide for a definite period, the remainder serving as a control. After the carbon dioxide treatment, the petioles and midribs were removed from the sample of leaves, and the juice was extracted from the blades. The juice was also extracted from the blades of the control leaves. The remainder of the carbon dioxide-treated leaves was left in the open for 1 hour, after which the juice was extracted from the blades. The pH was determined on the three lots of juice. Each lot was also analyzed for total soluble, ammonia, and amide nitrogen as outlined by Nightingale, Robbins, and Schermerhorn (2). The changes in pH, ammonia, and amide fractions resulting from the carbon dioxide treatment are shown in Table III.

No significant change in the water-soluble nitrogen (that fraction which is not coagulated by acid or heat) was found to occur in the plants during treatment. During the exposure to high concen- trations of carbon dioxide, ammonia was split off from certain compounds in sufficient quantities to account in full for the increase in pH. It is evident from the data that ammonia is formed partly at the expense of the acid amides in all experiments. In Experiment 1 of Table III, the change in pH due to the treat- ment was from 6.00 to 7.20. The ammonia nitrogen increased from 3.52 to 11.00 per cent, or a net increase of 7.48 per cent. At the same time the amide nitrogen decreased from 16.43 to 8.50 per cent, a net decrease of 7.93 per cent. In other words, the decrease in amide nitrogen accounts in full for the ammonia formed in Experiment 1. Calculations show that the amount of ammonia formed will account in full for the decrease in the hydrogen ion activity observed. The data show that acid amides are hydro-

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652 Nitrogen of Sugar-Beet

lyzed with the liberation of ammonia. The following equation suggests the reaction probably involved: 2R. CONHz + 3Hz0 yields R. COONH4 + R. COOH + NH40H. From the other two experiments, however, it is evident that ammonia is being split off from compounds other than acid amides. In Experiment 3, where the carbon dioxide concentration was 80 per cent, only 10 per cent of the ammonia came from the acid amides. This shows that the type of compounds furnishing the ammonia depends on

TABLE III

Changes in pH, Ammonia, and Amide Nitrogen of Sugar-Beet Leaves Due to

EXpS& ment NO.

Blades from plants

1

2

3

Untreated

Treated Untreated Treated

‘I recovered* Untreated

Treated ‘I recovered*

CO2 Treatment

N in HTO- Treatment soluble

PH fraction

Gftin

(dry bask) N&

1 Time NH2 (Amide - CO2

-

Per cent

-I. min.

20 90

40 60

40 60

80 90

80 90

-- ---A

Per Pm Pet Per Per cent cent cent cent cent

6.00 3.52 16.43 7.2011.00 8.50 7.48 7.93 106 6.34 2.02 5.82 7.27 7.00 3.63 4.98 2.20 44 6.62 3.75 4.48 6.61 2.59 1.56 7.06 8.22 0.98 5.63 0.58 10

6.43 3.69 1.42

* These plants were allowed to remain in the laboratory only 1 hour be- fore being extracted. According to results shown in Fig. 1, it requires a longer period for the plants to return to normal. It is quite probable

that the ammonia and amide nitrogen would have returned to normal if a longer recovery period had been allowed.

the concentration of carbon dioxide to which the plants are sub- jected. When the plants were allowed to recover from the carbon dioxide treatment, the ammonium salts were reconverted to the acid amides and other compounds which were affected by the treatment with a production of hydrogen ions. Previous experi- ments indicated that if the carbon dioxide had been allowed to stand longer before the juice was expressed, the pH, and amide and ammonia nitrogen would have returned to normal.

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J. M. Fife and V. L. Frampton 653

DISCUSSION

Certain plants, particularly sugar-beet, when subjected to a high concentration of carbon dioxide, catalyze certain reactions at a very rapid rate, which counteract an increase in the hydrogen ion activity of the cell sap. One of the catalytic reactions which apparently takes place in the beet plant, and which neutralizes the hydrogen ions formed, is as follows: R.CONH2 + 2Hz0 yields R. COOH + NHd+ + OH-. The concentration of hydroxyl ions produced will depend on how far the reaction proceeds and the strength of the organic acids liberated. A survey of the disso- ciation constants of the different organic acids revealed that the amides of the amino acids must furnish a large portion of the ammonia to account for the increase in pH by the above reaction. It is evident from Table III, however, that ammonia is split off from other soluble compounds which may not be acidic in nature, such as free amino groups of soluble proteins.

This would account for the maximum increase in pH observed (Fig. 2) when the plant is subjected to 80 per cent carbon dioxide. When the plant is subjected to 100 per cent carbon dioxide, two of the reactions occurring in the leaf tend to counteract each other. In the first reaction, the plant produces hydroxyl ions which neutralize the carbonic acid in the cell sap. Where the oxygen tension is extremely low, comparatively strong organic acids form an important part of the end-products of respiration. These acids formed in respiration would neutralize the hydroxyl ions formed in the first reaction. Consequently, the pH of plants treated with 100 per cent carbon dioxide would depend on the length of time the plants were exposed to the gas. If the exposure is relatively short (the plants removed before the free oxygen is completely exhausted), the pH of the extracted juice would be greater than that of untreated plants. If, however, the plants are exposed to the pure gas for a long period, the organic acids formed in respiration would mask the former reaction and cause the hydro- gen ion concentration of the cell sap to become higher than normal.

When carbon dioxide-treated beet plants or leaves are allowed to recover, the amide nitrogen increases to its original value apparently at the expense of the ammonia nitrogen with the formation of hydrogen ions. The reaction may be shown as follows: R. COOH + NH4+ yields R. CONHz + Hz0 + H+.

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654 Nitrogen of Sugar-Beet

It is well known that the juice from the leaves of such plants as the sugar-beet, tomato, Chenopodium murale, and others, is very poorly buffered at their normal pH. Despite the extremely low buffer index of the juice of the beet leaf (0.0048 between pH 6 and 7), the plant is able to maintain its normal pH when subjected to extremely adverse conditions. It seems logical to suppose that the plant complex is so arranged that the normal pH could be maintained by means other than the buffer capacity of the cell sap. The above reactions apparently accomplish this purpose in the beet plant, for the reaction proceeds in the beet plant in either direction at a very rapid rate, producing hydrogen or hydroxyl ions according to the conditions imposed.

It is possible that further studies on the reactions catalyzed by the beet plant when under the influence of high concentrations of carbon dioxide might shed light on the reactions involved in the formation of amides, amino acids, and proteins in plants.

SUMMARY

Analyses of juice expressed from sugar-beet plants immediately after treatment with high concentrations of carbon dioxide show that striking chemical changes have taken place in the cell sap of the tissues.

Certain reactions which are catalyzed by the beet plants, exposed to high concentrations of carbon dioxide, prevent enormous increases in the hydrogen ion concentration of the cell sap. When the juice is expressed from the beet plant immediately after treat- ment with carbon dioxide, a determination of the increase in pH over that of normal juice indicates to what extent these reactions took place.

When the beet plants are exposed to a high concentration of carbon dioxide, ammonia is split off from acid amides presumably according to the reaction, 2R. CONH2 + 3Hz0 yields R. COONH, + R. COOH + NHd+ + OH-.

The increase in ammonia nitrogen found in the juice accounts in full for the observed increase in pH. When high concentrations of carbon dioxide were applied to the plant, ammonia nitrogen was found to have been split off from soluble nitrogenous compounds other than the acid amides.

Beet plants respond rapidly to carbon dioxide treatment. A

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J. M. Fife and V. L. Frampton

significant increase in pH of the extracted juice was obtained after 5 minutes exposure to the gas, and a maximum pH was reached in approximately 1 hour. Recovery from carbon dioxide treatment (a return to initial pH) was found to be almost as rapid. The beet plants recovered in about 2 hours after removal of the plants from the gas chamber.

The juice expressed from carbon dioxide-treated beet plants is stable with respect to pH. The catalytic agents which accelerate the reaction or reverse the process are active only in the organized plant. This increase in pH as a response to carbon dioxide expo- sures appears to be a general type of response, for ten other species of plants were found to respond in a similar manner.

BIBLIOGRAPHY

1. Magness, J. R., and Diehl, H. C., J. Agric. Research, 27, 1 (1924). 2. Nightingale, G. T., Robbins, W. R., and Schermerhorn, L. G., New

Jersey Agric. Exp. Stat., Bull. 48 (1927). 3. Robertson, G. R., Ind. and Eng. Chem., Anal. Ed., 3, 5 (1931). 4. Thornton, N. C., Contrib. Boyce Thompson Inst., 6, 403 (1933). 5. Willaman, J. J., and Beaumont, J. H., Plant Physiol., 3, 45 (1928).

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J. M. Fife and V. L. FramptonSUGAR-BEET PLANT

NITROGEN FRACTIONS OF THEUPON THE pH AND CERTAIN

THE EFFECT OF CARBON DIOXIDE

1935, 109:643-655.J. Biol. Chem. 

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