THE SOURCE OF AMMONIA IN PLANT TISSUE · PDF file716 AMMONIA IN PLANT TISSUE EXTRACTS. II...

THE SOURCE OF AMMONIA IN PLANT TISSUE EXTRACTS

II. THE INFLUENCE OF UREA

BY I. REIFER AND J. MELVILLE

(From the Plant Chemistry Laboratory, Department of Scientific and Industrial Research, Palmerston North, New Zealand)

(Received for publication, December 22, 1948)

In a previous paper (1) we have drawn attention to the discrepancy in free ammonia values of fresh and dried green leaf tissue. The evidence was derived largely from the pasture grasses but the same observation was made with other species; viz., that in practically all cases the ammonia of dried tissue was significantly higher than that of fresh tissue. It was shown that the increase in ammonia due to drying could not be attrib- uted to the hydrolysis of glutamine in the oven, and we tentatively postulated the presence in leaves of an ammonia precursor which is even more heat-labile than glutamine.

Attempts were then made by infiltration techniques to increase the concentration of this ammonia precursor as measured by the difference between fresh and dried ammonia values. A wide variety of nitrogenous compounds was introduced into the leaf and it was found that urea caused the largest increase in the concentration of ammonia precursor. The possibility that urea was responsible for the increase in ammonia on drying was therefore investigated and in this paper are recorded certain observations on the metabolism of urea in plants.

EXPERIMENTAL

Methods for the preparation of plant extracts and analytical procedures for the estimation of ammonia and glutamine are as described in the previous paper (1). Urea was determined by means of urease, the resultant ammonia being distilled in vacua and estimated by hypobromite oxidation. Jack bean meal, commercial samples of urease, and crystalline urease were used at various times throughout the investigation.

Urea determinations have been made on some hundreds of samples of grasses under a wide variety of environmental conditions and on a limited range of other plants, including peas, beans, silver beet, clovers, tomatoes, etc., under ordinary field conditions. Values between 1 and 5 mg. of urea N per 100 gm. of fresh tissue were usually obtained, the values being almost always higher than those for free ammonia and of the same order as those for glutamine. Rye-grass plants, adequately fertilized with ammonium sulfate, have given values up to 15 mg. per cent of urea N under

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716 AMMONIA IN PLANT TISSUE EXTRACTS. II

certain environmental conditions. On the other hand, a few samples of the same species over the past 2 years have been found to contain no measurable quantity of urea, and it is noteworthy that a proportion of these samples showed little or no increase in ammonia when dried.

In order to explore the possibility that urea was responsible in whole or in part for this increase, the behavior of tissues artificially enriched with urea was studied. Infiltration as a method of enrichment was used in earlier experiments but was later replaced either by culture of detached leaves in dilute urea solution (0.1 to 1.0 per cent) or by heavy fertilization of intact plants in the field (5 to 10 gm. per plant watered into the soil). Uptake of urea was of course rapid in detached leaf cultures, but the speed with which urea absorption by the roots was manifested in the leaves was somewhat surprising. For example, a rye-grass plant before fertilization contained 1 mg. per cent of urea N in the leaves; within 1

TABLE I Effect of Culture of Rye-Grass Leaves in 0.6 Per Cent Urea Solutions on NHa-N and

Glutamine Amide N, Expressed As Mg. per 100 Gm. Fresh Weight -

I Ammonia N Pe.iod of culture I -------.--.-..-~--

Fresh tissue Dried tissue Fresh tissue

hrs

0 1 2 4 6

24

0.7 1.4 3.6 1.40 8.5 10.4 0.47 14.4 25.8 1.05 14.6 37.2 2.68 12.5 44.7 0.47 23.3 46.5

-

Glutamine amide N

hour the value had increased to 4 mg. per cent and the concentration reached a peak of 35 mg. per cent of the fresh weight between 16 and 24 hours. In culture experiments, values for urea N of 45 mg. per cent have been recorded after 16 hours.

Behavior of Urea-Enriched Tissues on Drying-When artificially enriched tissues are rapidly dried at 80”, the increase in ammonia on drying is very much greater than in any case cited in the previous paper. In Table I are given typical results of ammonia values for fresh and dried rye-grass leaves after culture in dilute urea solutions, and parallel glutamine values are included to illustrate the rapid and consistent increase in this metabolite under these conditions. The figures demonstrate the rapidity with which urea is absorbed, the consistently low values for free ammonia in the fresh leaves, and the large divergence between the ammonia values of fresh and dried tissue after the 1st hour of culture.


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1. REIFER AND J. MELVILLE 717

These results, together with the demonstration that urea is a normal leaf constituent, offer strong presumptive evidence for the hypothesis that urea is the source of the extra ammonia found on drying. The breakdown of urea cannot, however, be explained on the grounds of heat lability, since urea is considerably more stable than glutamine when heated in aqueous solution. An alternative explanation is that the breakdown is enzymatic, and the following evidence is adduced to show that this explanation is reasonable.

The establishment of urease as a normal constituent of leaf tissue was the first requirement, and we have confirmed the findings of earlier workers that urease is widely distributed t,hroughout the plant kingdom. By incubation of fresh leaf macerates with urea in the presence of H& for periods up to 1 hour at 37”, every tissue examined has shown considerable urease activity.

It then remained to show that the conditions of drying used in this Laboratory, viz. dehydration to 5 per cent moisture level at 80” f 2” in 35 to 45 minutes, are such that the hydrolysis of urea is possible. From careful study of the tissue during the drying period, it is believed that the course of events is as follows: the stream of air at 80” heats the tissue quite rapidly to a temperature (probably about 60”) at which the cell structure begins to break down; dehydration of the cytolyzing leaves is rapid and the latent heat of vaporization prevents any marked rise of local temperature until drying is almost complete. The conditions during this period of cytolysis at intermediate temperatures are conducive to rapid hydrolysis of urea without too serious a loss of urease through heat inactivation.

Two lines of evidence support this general postulate. The first comes from drying experiments at 50”, at which point there is no obvious cytolysis, and at 80” on enriched leaves which were infiltrated with mercuric chloride to prevent enzyme action. In a typical example, the ammonia values for fresh leaves, leaves dried at 80” (for 35 minutes), leaves dried at 80” in the presence of mercuric chloride, and leaves dried at 50” were 1.23, 6.54, 2.37, and 3.21 mg. per cent respectively. When leaves were cytolyzed with ether before drying, the results at 50” and 80” were practically identical.

The second line of evidence is that leaf tissue dried at 80” still exhibits considerable urease activity, as is demonstrated in the following experi- ment. Dried and ground leaf tissue containing only 1.0 mg. per cent of urea N was incubated with dilute urea solution for 30 minutes at 37” with a consequent increase in free ammonia N of 4 mg. per cent. The demonstration that tissue urease is not completely destroyed during drying has a further implication with respect to the standard extraction pro-


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cedure. Normally the ground tissue is extracted with water at 80” for 10 minutes (2). We felt that, if the urease survived drying at 80”, further hydrolysis during extraction with hot water might occur. A comparison was, therefore made between extraction of dried tissue in the War- ing blendor in the cold, and by the standard hot water procedure, the relevant results being shown in Table II. Also included are the fresh ammonia values and the results of further incubation for 1 hour at 37” of the extracts prepared at 80” to show that inactivation is still not complete. The freeze-dried tissue in which presumably no inactivation of enzyme took place during drying shows large increases in ammonia, both on extraction and subsequent incubation of the hot water extract.

It is reasonable to assume from these data that urea can be enzymati- tally hydrolyzed during drying, and, on this hypothesis, it is apparent that the level of ammonia in a dried tissue which contains urea and which is

TABLE II Production of Ammonia from Extracts of Urea-Enriched Tissues, Expressed As Mg.

per 100 Gm. Fresh Weight

Treatment of tissue Fresh tissue Cold water Hot water

extract extract Hot water

extract incubated

1% urea culture for 16 hrs.. . . . . . . . . . 0.70 7.5 18.4 22.5 Freeze-dried, 1% urea culture, 16 hrs.. . . . . . 0.12 7.35 21.4 0.5% urea culture, 16 hrs.. . . . . . . . . . . . . 1.28 3.1 9.58 14.7 Fertilized plant, 16 hrs.. . . . . . . . . . . . . . . . . 0.70 1.90 4.2 6.4

T

-

Dried tissue

extracted at 80” is the result of at least three factors, viz. the concentration of ammonia in the fresh leaf, and the hydrolysis of part of the urea in the oven and of a further part during extraction with hot water.

The finding that tissue urease is not inactivated by heating for 10 minutes at 80” in dilute aqueous solution is not in accord with the generally accepted properties of enzymes. In this connection we have found that, although an aqueous solution of jack bean urease lost its activity completely under these conditions, some potency still remained when heating was carried out in the presence of urea. It may also be assumed that the tissue colloids exerted some degree of protection during drying and hot water extraction.

E$ect of Cytolysis and Incubation on Urea-Enriched Tissue-Further insight into the urea-m-ease system in leaves is gained from the behavior of macerated and cytolyzed leaves on incubation. When tissues are macerated in the Waring blendor without any additions other than ice


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I. REIFER AND J. MELVILLE 719

and water, the resultant macerate shows little or no urease activity, presumably because of inactivation by oxidation with atmospheric oxygen. This phenomenon is also shown by jack bean urease. A partial reactiva- tion occurs on standing at room temperatures, and production of ammonia by urea-containing tissue occurs slowly after the 2nd hour. If, however, a reducing agent such as H2S is added during blending, inactivation is prevented and the free ammonia value begins to rise immediately. Cytolyzed leaves behave in a fashion similar to that of macerates in the presence of a reducing agent and the results of incubation of cytolyzed and macerated leaves under a variety of conditions are given in Table III. They may be summarized as follows: (1) Leaf urease is seriously but not quantitatively inactivated by blending, inactivation being pre-

TABLE III Ammonia N of Macerated and Cytolyzed Leaves after Standing for 16 HOUTS at Room

Temperature, Expressed As Mg. per 100 Cm. Fresh Weight Proce~

1 Waring blendor macerate 6.55 2 Macerate with acetic acid to pH 4.5 1.22 3 Macerate with H,S 26.9 4 Ether-cytolyeed tissue 24.3 5 Cytolyzed tissue with acetic acid 3.56 6 Tissue boiled 3 min. 2.04 7 Equal volumes of (6) and (4) 45.9 8 Cytolyzed tissue + urea (= 12.0 mg. % N) 35.9 9 As (8), but with acetic acid 3.9

10 Tissue boiled 3 min. and urease added 22.5

Treatment of tissue Tissue 1 Tissue 2

11.9 2.34

27.8 26.0 3.21 3.1

2.5 24.0

- zaallated

48.6 36.3

vented by addition of reducing compounds. (2) Cytolyzed leaves and reduced macerates give substantially the same ammonia values on standing for 16 hours. (3) Leaf urease, like jack bean urease, is practically in- active at pH 4.5. (4) Added urea is quantitatively hydrolyzed by cytolyzed leaves and by reduced macerates, while the addition of urease to a heat-inactivated tissue gives substantially the same ammonia value after 16 hours as is given by the same tissue on cytolysis and incubation.

The results offer a reasonable explanation for data reported in the previous paper, where a comparison was made between extracts prepared by cytolysis and pressing and those prepared by blending. The results were consistently higher for the former technique, and it is obvious that at least a partial explanation is the hydrolysis of tissue urea which occurs during the time required for preparation of the extract by the cytolysis method. The results also support the conclusion that of the methods


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available for estimation of free ammonia in leaf tissue the most accurate is rapid maceration of fresh tissue in the cold at pH 4.5.

Presence in Leaves of Ammonia Precursors Other Than Urea-The results presented thus far are designed to show the presence in leaves of urea and urease, to describe the behavior of leaf tissue containing large concentrations of urea under various conditions, and, on the basis of the similarity in behavior between urea-enriched and normal leaves, to offer the explanation that the observed increase in ammonia on drying is due in part to the enzymatic hydrolysis of urea in the oven. Ammonia increases, however, cannot be wholly explained in this way. When urea determinations are made concurrently with ammonia determinations on fresh and dried tissue, it is immediately apparent that other potential ammonia precursors are present. From the hundreds of samples which have been analyzed in an attempt to characterize these materials, we have collected in Table IV a series which best demonst,rates the effect.

The characteristic feature of the data is that the sum of ammonia and urea nitrogen for dried is higher than that for fresh tissue. Large increases of ammonia invariably occur (cf. Tables I to III), while there is a con- current increase in urea in all the samples listed. If urea were the sole source of ammonia increase on drying, the rise in ammonia would be bal- anced by a corresponding decrease in urea. Table IV also provides further evidence for the conclusion in the previous paper that glutamine breakdown cannot account for the discrepancy. It is necessary therefore to postulate a source of ammonia other than urea, and initially it was difli- cult to decide whether the unknown material was breaking down to ammonia directly or by way of urea. From Table IV and from a large col- lection of other data, it appears probable that leaves contain a material which yields urea, and we shall refer to this material in the remainder of this paper as the “urea precursor.” The effect is particularly well demonstrated in Tissues 5 and 6 of Table IV, where the ammonia of the dried tissue is considerably greater than the ammonia plus urea of the fresh tissue. Our conclusion is that in these cases the breakdown of precursor in the oven is more important than the hydrolysis of the preformed urea. Tissue 7 is included to show the essentially similar behavior of normal tissue.

Additional information on this point comes from utilization of the observed inactivation of tissue urease which occurs in the Waring blendor. Extracts of fresh tissue were prepared by blending in the presence and ab- sence of HzS, and analyses were made for ammonia and urea after incubation for 5 hours at 37”. The results are shown in Table V and again it will be noted that the normal tissue behaves in essentially the same way as artificially enriched tissue.


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The trend is the same in every case. When H&3 is added before maceration, the ammonia increases markedly on incubation. When H2S is omitted, the increase is significant, due probably to partial reactiva- tion of the oxidized enzyme on standing, but is very much smaller than when the enzyme is protected from inactivation. The figures in Column 4 are consistently higher than those in Column 6, and we believe that the

TABLE IV Comparison of Ammonia, Urea, and Glutamine Amide N on Fresh and Dried Rye-Grass

Extracts, Expressed As Mg. per 100 Gm. Fresh Tissue

Tissue No.

1. 1% urea culture, 16 hrs ......... 2. 1% ‘I “ 16 “ ........ 3. 0.5% urea culture, 16 hrs ....... 4. 1% urea culture, 16 hrs ......... 5. Fertilized plant, 16 hrs .......... 6. Same,64 hrs .................... 7. Normal leaves ..................

. . .

T

~

I

-

Fresh tissue extract Dried tissue extract

YHa-N

0.70 0.23 2.45 1.40 3.98 0.47 1.11

-

IT. 18.7 20.8 11.8 38.2 19.0 31.2 10.9 35.4 12.6 17.1 8.7 12.8 12.0 31.9 26.2 4.9* 43.7* 27.4% 2.44 12.1 11.7 20.2 15.7 0.93 22.9 3.8 3.2 17.1 1.63 3.16 1.39

- * This tissue was freeze-dried and extracted at 80” for 10 minutes.

TABLE V Effect of Incubation of Fresh Rye-Grass Extracts on Ammonia N and Urea N,

Expressed As Mg. per 100 Gm. Fresh Tissue --

Fresh extract by3b;y2

Waring blendor Waring blendor macerate, incubated macfxate +

Tissue for 5 hrs. Hr;;F$$ed

_.____~~ NHa-N UreaN NHa-N Urea N NHrN UreaN

(1) (4) ___- p2_ (3) -~~-- ~ (5)- . .._(q

Urea fertilization, 18 hrs.. . . . . . . . . . 1.23 5.24 2.45 9.75 7.06 5.64 Same .__..__..._......__............... 0.76 4.94 2.10 10.03 5.78 6.46 h’o treatment.. 0.79 1.78 2.16 I 2.69 2.86 1.69 0.1% culture, 16 hrs.. . . 1.11 7.41 , 2.68 / 12.50 6.77 6.83 -. --______ .-__ -..----

most probable explanation is that the urea precursor is hydrolyzed enzymatically to urea, and that where urease is inactivated by aeration (Col- umn 4) marked urea formation takes place. This explanation appears to cover the observed facts on drying and incubation, and future work is being planned on the basis that the unknown material breaks down through urea to ammonia.


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722 AMMONLA IN PLANT TISSUE EXTRACTS. II

On current theories of urea metabolism, the most probable urea precursor in any organism is arginine, and several attempts have been made, by enzymatic and calorimetric methods, to demonstrate decreases of arginine with increases either of urea or ammonia. No correlation has been obtained; arginine values remained relatively constant during the procedures outlined above.

The figures in Column 6 of Table V are of interest from another point of view. From the ease and rapidity with which added urea is broken down by cytolyzed leaves and by reduced macerates, it would be expected that no urea would be present after 5 hours incubation; i.e., that the values in Column 6 would be zero. This is never the case, while an exactly parallel observation has been made on many plant extracts which have been incubated with jack bean urease. For example, a tissue containing originally 0.24 mg. of ammonia N and 5.76 mg. of urea N per cent was macerated in the presence of H&3 and incubated with urease for 2 hours, when the corresponding figures were 1.81 and 4.44 mg. per cent respectively. Thus, despite the presence of relatively excessive amounts of urease during the period of incubation, the addition of fresh urease for the residual urea determination has brought about further production of ammonia. Moreover, when urea was added to the macerate in quanti- ties greater than that present in the tissue, it was quantitatively recovered as ammonia after 2 hours. This last finding might be explicable on the ground that jack bean meal contains amidases other than urease, were it not for the observation that crystalline urease behaves in the same man- ner. No explanation can be offered for this anomalous behavior, which is reported to emphasize the complexity of the systems leading to the production of ammonia in green leaves.

Efect of Environmental ConditionsIt has already been mentioned that the phenomena under investigation, which hinge around urea and related compounds, manifest themselves in greatly varying degree throughout the year. There are seasons of the year when no urea or urea precursor can be found in rye-grass and when there is no significant increase in ammonia when the tissue is dried. At certain times of the year, heavy fertilization of rye-grass plants with urea causes little or no rise in urea concentration and no apparent production of urea precursor, even though there is a rapid increase in other soluble nitrogenous components, indicating that the urea is being absorbed. Moreover, if leaves from similar plants are cultured in dilute urea solutions, the leaf urea level rises as usual but there is no production of urea precursor. The change-over from one type of utilization mechanism to the other is illustrated in Table VI, where two apparently identical rye-grass plants derived from the same parent clan were heavily fertilized with urea on October 27 and November 1 respectively, and analyzed on October 28 and November 2. It can be seen that


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within the short space of 5 days a very different picture is obtained. This is not an isolated finding, since no tissue analyzed between July and about the end of October showed any significant production of urea precursor under urea culture or fertilization, whereas from November unt.il April the majority of rye-grass plants behave similarly to the second one shown in Table VI. This latter generalization holds true, except under dry hot conditions which may cause cessation of growth during January and Feb- ruary, and before the autumn rains, which in New Zealand normally fall during March.

The rate of growth is certainly not the governing factor in the change from one type of metabolism to the other, since rye-grass under our conditions grows at least as rapidly during August and September as in No- vember. From our observations for over nearly 3 years, we have come to the conclusion that urea metabolism follows substantially different path-

TABLE VI

Dij’erential Response of Rye-Grass Plants to Urea Fertilization

The results are expressed as mg. per 100 gm. of fresh tissue. --.

Fresh tissue Dried tissue

1948

AmmNOnia Urea N AwnNOnia Urea N -.-.____ __- --

Oet. 28.. . . . . . . . . . 0.62 1.36 3.15 1.23 Nov.2..................................... 0.64 19.6 6.25 31.5

ways at different seasons of the year, which is equivalent to concluding that the enzymatic complex is subject to considerable variation.

DISCUSSION

The high levels of urease in certain seeds, established between 30 and 40 years ago, together with subsequent investigations showing the wide distribution of urease throughout the plant kingdom, have naturally led to speculation about the part played by the urea-urease system in plant metabolism. The presence of urea in the vegetative portions of higher plants was first satisfactorily shown by Fosse (3), but, through his use of the non-specific xanthydrol reagent, he was unable to distinguishbetween free and combined urea. Klein and Taubock (4), using the urease method, were able to show beyond doubt that free urea occurs in higher plants, although they were able to demonstrate its presence only in actively metabolizing tissues. From numerous experiments on seedlings grown in solutions of arginine, Klein and Taubock concluded that the greater part of the urea of higher plants results from the hydrolysis of arginine.

Since these publications, the last of which appeared in 1933, we have


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found only one paper dealing with urea concentrations and urea metabolism in plants, viz. that of Damodaran and Venkatesan (5), which appeared while this paper was in course of preparation. These investiga- tors showed that, during the germination of Dolichos biji!orus and Phaseo- lus mungo, urea concentrations rose sharply and reached high levels between the 10th and 20th days. Simultaneous arginine determinations led to the conclusion that only part of the urea could be accounted for by decrease in arginine. Apart from this paper, no attention appears to have been paid to urea during the past 15 years and the reviews of the nitrogenous metabolism in plants published during that period make only passing reference to urea, the whole emphasis in ammonia metabolism being placed on asparagine and glutamine.

It is not suggested that the data presented in this paper in any way in- validate the conclusions reached by Vickery, Chibnall, and their co- workers as to the key position of asparagine and glutamine in plant nitrogen metabolism; but the demonstration both in this paper and by Damodaran and Venkatesan of the presence in plants of urea justifies its inclusion as a third amide in the nitrogen cycle.

No specific information can be offered as to the part played by urea in nitrogen metabolism. The rapid uptake of urea by intact plants through the roots and the utilization of this urea to form glutamine may mean nothing more than that, if urea is absorbed by the plant, the urea- urease system can act as an internal source of ammonia. On the other hand absorption through the roots does not explain the presence of urea to the extent of 1 to 5 mg. per cent in a wide variety of plants which have received no urea fertilization; nor does it explain the fact that rye-grass well fertilized with ammonium sulfate may contain up to 15 mg. per cent of urea under suitable environmental conditions. The observed phenomena may be explicable on the assumption of the operation of a Krebs ornithine cycle, in which case urea would arise from hydrolysis of arginine. Free arginine exists in the plant sap, and, even though the concentration is low,’ there is a large reserve in the leaf protein which could be drawn on as required.

That this is not the complete explanation is seen from Tables IV and V, as well as from the studies of Fosse and of Klein and Taubock. The evidence is based largely on the behavior of artificially enriched leaves, but it must be stressed that the purpose of enrichment was solely to exag- gerate and define clearly an effect which is also given by normal leaves. There is no reason to suppose that normal leaves do not follow the same metabolic course at a lower level.

Serious consideration does not appear to have been given to the possi-

1 Bathust, N. O., private communication.


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bility that urea may act as a raw material for synthesis in biological systems without the necessity of first being hydrolyzed to ammonia. Two lines of evidence point to this possibility in plants. Firstly, the urea precursor reaches higher levels under urea culture and fertilization than are reached when ammonia is used. Secondly, there are consistent and, we believe, significant differences in glutamine values for plants which re- ceive their nitrogen as urea and ammonia respectively. If urea were acting only as a source of ammonia, it would be expected that no differences would be apparent in either case.

The postulate that the increase in ammonia on drying comes from the hydrolysis in the oven of urea and of a urea precursor is at variance with the conclusion reached in the previous paper (1); viz., that there exists in leaves an ammonia precursor which is more labile than glutamine. Since our recognition of the possibility of enzymatic activity during drying, no evidence for a heat-labile ammonia precursor has been obtained and the earlier conclusion is therefore withdrawn.

Apart from the importance of the establishment of urea as a plant metabolite, there is a further point of interest in studies of heavy urea fertilization. Animal production in New Zealand is almost entirely based on pastures, and investigations of the effect of the grazing animal on the growth of pasture have been in progress for a number of years (6). It has been shown that grazing animals return up to 500 pounds of nitrogen per acre in dung and urine, and that of this amount over 50 per cent is in the form of urea. The concentration of urea in a single urine patch is high and an investigation is in progress as to the proportion of this urea which is absorbed as such by the pasture plants, the proportion which is absorbed as ammonia arising from hydrolysis of the urea, and the proportion which is oxidized to nitrate and absorbed in that form.

The technical assistance of Miss J. L. Fisher and Miss R. N. Haycock is gratefully acknowledged. Thanks are also due to Dr. J. B. Sumner for gifts of crystalline urease and of jack beans, and to the Director and staff of the Grasslands Division, who have supplied the raw material on which this investigation was based.

SUMMARY

Urea and urease are present in significant concentrations in a series of plants, and the former hydrolyzes enzymatically during drying at 80” to give ammonia. Evidence is also presented for the presence under certain environmental conditions of a urea precursor which hydrolyzes to give first urea and finally ammonia when leaves are dried in the oven or incubated after cytolysis with ether.


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BIBLIOGRAPHY

1. Reifer, I., and Melville, J., Tr. XI Internat. Congr. Pure and Appl. Chem., in press.

2. Vickery, H. B., Pucher, G. W., Clark, H. E., Chibnall, A. C., and Westall, R. G., Biochem. J., 29, 2710 (1935).

3. Fosse, R., L’uree, Paris (1928). 4. Klein, G., and Taubock, K., in Klein, G., Handbuch der Pflanzenanalyse, Vienna,

4, pt. 1, 197 f f . (1933). 5. Damodaran, M., and Venkatesan, T. R., Proc. Indian Acad. SC., 27B, 26 (1948). 6. Sears, P. D., and Newbold, R. P., New Zealand J. SC. and Tech., 24A, 36 (1942).


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I. Reifer and J. MelvilleINFLUENCE OF UREA

TISSUE EXTRACTS: II. THE THE SOURCE OF AMMONIA IN PLANT

1949, 178:715-726.J. Biol. Chem.

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