THE ASSIMILATION OF AMMONIA NITROGEN BY THE TOBACCO PLANT: A PRELIMINARY STUDY WITH ISOTOPIC NITROGEN BY HUBERT BRADFORD VICKERY AND GEORGE W. PUCHER (Prom the Biochemical Laboratory of the Connecticut Agricultural Experiment Station, New Haven).4ND RUDOLF SCHOENHEIMER AND D. RITTENBERG (Prom the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York) (Received for publication, July 3, 1940) When ammonium chloride that contains an excess of nitrogen of atomic weight 15 is administered to plants grown in culture solution, it is possible, by means of isot,opr analysis of suitable fractions of the tissues, to detect certain details of the course of assimilation of this nitrogen. Reactions that arc roughly divisible into two categories are to be anticipated; first, those that represent normal increase of tissue components associated with growth and, second, t,hose that represent interactions between substances already present and substances that contain the newly acquired nitrogen whereby the isotope is introduced into the former. Reactions of this second type, which result in a shift, of nitrogen from one compound to another, have been studied in mature animals in nitrogen equilibrium. The prompt introduction of isotopic nitrogen into many of the amino acids of the body proteins has been observed when isotopic ammonium salts or amino acids are administered in the food of such animals (1,2). The replacement of the protein nitrogen by the dietary nitrogen is clearly a result of continuous chemical reactions in which the body proteins are normally concerned. The demonstration of this type of chemical change is of especial importance, since it represents a form of metabolic activity the existence of which has only recently become appreciated in animals and of which there are few if any records in plants. 531
532 Assimilation of Ammonia by Tobacco Plants are less favorable objects for this demonstration than animals. At maturity, when the reproductive cycle is completed, senescence rapidly follows; with herbs, death of the entire plant may occur or, in other species, there may be a withdrawal of metabolic products into the root system for storage against the growth of the following season. A protracted period during which the situation is analogous to that of a mature animal in nitrogen equilibrium is not usually attained, since plants do not excrete nitrogen but respond to its administration by growth or by the development of the reproductive organs. The introduction of nut>riment int,o the plant system also presents problems somewhat different from those experienced with the animal. The greater part of the normal nutriment absorbed by the roots consists of inorganic ions. In the absence of suitable salts, growth diminishes sharply or ceases, and a condition is rapidly set up in which the plant utilizes its stores of inorganic material, nitrogen compounds, organic acids, and carbohydrates in ways different from the normal. Nevertheless it has seemed desirable to test t,he behavior of plants to which isotopic ammonium ions were administered through the roots in order to provide an indication of the types of reaction that occur. It was found that this nitrogen is rapidly absorbed and transported to all parts of the plant. Within a short time, the isotope could bc detected in the amide groups of asparagine and glutamine and also in the water-soluble tissue components that are precipitated by mercuric acetate and sodium carbonate (Neuberg reagent) ; that is, in the fraction that includes free amino acids and certain basic substances. In addition, considerable amounts of isotopic nitrogen were found in the proteins of the leaves, stalk, and root. The picture presented is one of great chemical reactivity, the amides and amino acids presumably behaving as intermediates in the synthesis of proteins from the newly acquired ammonia. EXPERIMENTAL Owing to the small supply of isotopic nitrogen available, experimentation was necessarily severely limited in scope. The 1 Plants are especially sensitive in their response to the administration of nitrogen; when any deficiency in the supply of nitrogen is made good, renewed growth is to be anticipated at almost any stage in the life history.
Vickery, Pucher, Schoenheimer, Rittenberg 533 culture solution employed had the composition KHzPOl 0.00325 M, Ca(NO& 0.00215 M, MgS04 0.00105 M, NH&l 0.0043 M, CaC12 0.00215 M. To this were added traces of boron and of heavy metals and 0.5 part per million of iron. The plants were grown according to the Shive technique, the solution being continuously renewed at such a rate that the effluent was maintained approximately at ph 5.0. For the experiment with isotopic nitrogen, a single tobacco plant41 days after transplantation of the seedling was TABLE Effect of Administration of Ammonium Chloride with 1.61 Atom Per Cent Nib Excess on Certain Nitrogenous Components of Tobacco Plant Tissues A single plant 41 days from the seedling stage was treated for 72 hours. The data are expressed as atom per cent Ni5 excess. Protein N... Ammonia N................ Amide N Water-soluble N pptd. by Neuberg reagent.. Water-soluble N not pptd. by Neuberg reagent.. Volatile base N.. Alcohol-soluble N not soluble in water.... Water-soluble N exclusive of ammonia N. Found Leaf I Calculated for c::t ;: in NH&l administered 0.099 a.2 0.260 21.5 0.217 17.9 0.184 15.2 0.123 10.2 0.030 2.5 Stalk 0.184 15.2 0.275 22.7 0.286 23.6 0.225 18.6 0.108 8.9 0.078 6.4 0.104 8.6 _- - Calcu-?Ep: 1 Found cent N s in NHGl administered 0.222 18.4 0.779 64.3 0.200 16.5 0.414 34.2 transferred to an otherwise similar culture solution in which ammonium chloride with 1.21 atom per cent N15 excess was substituted for the ordinary ammonium chloride; 3 days later the plant was dissected into leaves, stalk, and roots and the tissues were weighed and dried at 80 for analysis. The analytical methods employed have been described in previous papers (3, 4) and the fractions prepared from each tissue are shown in Table I. Table I gives the results of the isotope analyses expressed in atom per cent N16 excess in each fraction and also the values cal-
534 Assimilation of Ammonia by Tobacco culated from these results that would have been obtained if 100 per cent isotopic nitrogen had been used instead of 1.21 atom per cent excess material. This calculation is in accordance with the method of presentation in other recent papers in which N16 has been used as a biological tracer element. Each figure gives directly the percentage of the. nitrogen that had been derived from the ammonia of the culture solution during the experimental period. The provision of control plants and of a sufficient number of experimental plants so that an accurate estimate could be made of the rate of growth of this plant under the experiment,al conditions was not possible. However, earlier studies of the rate of growth of tobacco plants in the field (3) have shown that individuals of t hc size and age of the present plant might be expected to have TABLE Protein h itrogwz in Tobacco Plant Treated with Isotopic Ammonia for 72 Hours I Total N Amount of N II 7n.q. microeq. Leaf protein N... 29.55 29.2 Stalk... 3.77 6.9 Root I... 5.32 11.8 Total... Administered NIL-N.... acquired approximately 25 per cent of their dry weight and nitrogen in the preceding 3 days. During the 72 hour experimental period, the plant absorbed 0.1498 gm. of ammonia nitrogen and 0.0737 gm. of nitrate nitrogen as determined from analysis of the culture solution. At the end, it contained 1.069 gm. of nitrogen of which 0.725 gm. was found in the leaves, 0.197 gm. in the stalk, and 0.147 gm. in the roots. Evidently, therefore, 20.9 per cent of the nitrogen had been absorbed during the previous 72 hours; of this the ammonia of the culture solution contributed two-thirds. A rough estimate of the rate of growth in the experimental period is thus provided. Table II shows the quantities of protein nitrogen, together with the amounts of isotopic nitrogen each tissue protein contained,
Vickery, Pucher, Schoenheimer, Rittenberg 535 calculated from the ratios in Table I. Since the ammonia nitrogen administered contained 1.21 atom per cent N15 excess, the whole plant had received 129 microequivalents of N15. The proteins had then acquired 47.9 microequivalents and, accordingly, 37 per cent of the absorbed isotopic nitrogen had found its way into the proteins of the tissues. This proportion is almost certainly significantly greater than could have been expected from the growth of new tissue and the synthesis of new protein during the experimental period, and strongly suggests the occurrence of chemical reactions by means of which proteins present from the start of the experimental period obtained isotopic nitrogen from other components of the cells. DISCUSSION Consideration of the data in Table I suggests that chemical reactions in which ammonia was concerned were especially marked in the roots. About 34 per cent of the water-soluble nitrogen, exclusive of ammonia, was derived from the ammonia of the nutrient solution during the experimental period. If this were due to the increase in number of organic molecules alone, it would be necessary to assume that one-third of this material had been developed within 72 hours. This is hardly likely and, as an altcrnative explanation, it may be suggested that reactions took place whereby newly acquired nitrogen was introduced into the watersoluble nitrogenous components of the cells. The high concentration of isotopic ammonia present in the roots at the end of the experimental period may also be pointed out in support of this view. The concentrations of isotopic nitrogen in the ammonia, the amide nitrogen, and the nitrogen of the Neuberg reagent precipitate (5) derived respectively from the stalk and leaves were substantially the same and of a magnitude not greatly different from that to be anticipated from the increase due to the growth of the plant. There is no clear evidence from these quantities alone that other reactions occurred by which nitrogen from the nutrient was introduced into compounds already present. The concentration of N15 in the nitrogen of the proteins of the stalk and leaves, particularly in the latter, was definitely lower than that observed in the ammonia and amide nitrogen of these
536 Assimilation of Ammonia by Tobacco respective tissues. It might be assumed from this that no reactions occurred in which the proteins shared, save the synthesis of protein due to growth. On the other hand, as is shown in Table II, when the actual quantities of protein are taken into consideration, appreciably more isotopic nitrogen was found in the proteins than can be reasonably accounted for in terms of growth; reactions that involve chemical interaction of nitrogen as between protein and soluble cell components apparently did occur. In order to provide clearer evidence of this, a sample of the protein of the leaf tissue was hydrolyzed and several amino acids were isolated. The methods employed were substantially those described by Schoenheimer, Ratner, and Rittenberg (2). Purified specimens of arginine, histidine, glutamic acid, and aspartic acid TABLE Ni6 Concentration (Atom Per Cent Excess) in Amino Acids and Amino Acid Fractions from Crude Protein of Leaves oj Tobacco Plant to Which Isotopic Nitrogen Had Been Administered Totalprotein... Arginine... Histidine... Glutamic acid... Aspartic. Copper salts insoluble in water and in methanol.. I I I I I but soluble in methanol. Remaining amino acids....... III 0.099 0.086 0.090 0.128 0.113 0.104 0.100 0.096 were secured for isotope analysis and, in addition, fractions of mixed amino acids obtained by the use of copper salts were examined. The data are given in Table III. The bases and the mixed amino acids in the copper salt fractions all had substantially the same proportion of isotope and this was not greatly different from that of the whole protein. This evidence alone would merely indicate the synthesis of new protein corresponding to the growth of the tissue. The glutamic and aspartic acids, however, contained a significantly larger proportion of isotopic nitrogen. To account for this in terms of growth alone would involve the unlikely assumption that the protein synthesized during the experimental period differed in amino acid composition from that present at the start in that it contained a much higher proportion of glu-
Vickery, Pucher, Schoenheimer, Rittenberg 537 tamic and aspartic acids. If the acquisition of isotopic nitrogen by the protein were solely due to growth, all the amino acids should have substantially the same isotope ratio, The augmented ratios observed for the glutamic and aspartic acids clearly imply that these amino acids interacted with other nitrogen or at different rates than the other amino acids and provide an example from plant tissues that is closely analogous to previous observations on the metabolism of proteins in animals (2). Some process in addition to mere growth of the plant with its attendant synthesis of protein must have occurred in order to account for the augmented replacement of the nitrogen of the glutamic and aspartic acids. This process must have been followed by the synthesis of these acids into the protein molecule and must have been preceded by the release of glutamic and aspartic acids or of their potential equivalents from protein already formed. The relatively low concentration of the isotope in the nitrogen of the substances that were not precipitated by the Neuberg reagent suggests that these components play a less active r81e in the nitrogen metabolism. The quantity of nitrogen was by no means inconsiderable, the order of magnitude being similar in each case to that of the nitrogen precipitated by the Neuberg reagent. Much of it, however, consisted of nitrate nitrogen which does not undergo spontaneous exchange with ammonia nitrogen (6). The nitrogen of the volatile base (exclusive of ammonia) in the leaf tissue, a part of which was identified as nicotine, contained very little isotope. A low order of reactivity of the nitrogen of this compound is suggested. That part of the nitrogen of the leaf and stalk insoluble in hot water but extracted by hot alcohol, a fraction which contained the chlorophyll, also showed only minor evidences of replacement. No attempt to account for the detailed mechanism whereby so large an amount of isotopic nitrogen was assimilated into the proteins of this plant can be advanced at the present time. However, it may be worth while to point out that, if the proteins of plant cells undergo continuous decomposition and resynthesis at a high rate, as is envisaged by the views of Gregory and Sen (7) on the mechanisms involved in plant tissue respiration, the rapid introduction of isotopic nitrogen into the proteins follows as a logical consequence.
53s Assimilation of Ammonia by Tobacco Buckwheat Experiment A similar experiment conducted with twenty-two buckwheat plants transferred for 47 hours, at the time of initial blossoming, to a culture solution identical with that employed for the tobacco plant experiment gave closely similar results. Some of the data have already been submitted in a preliminary communication (8). Owing to the small quantities of tissue available, the full data were less satisfactory than those from the tobacco plant and are therefore not given in detail. Isotopic nitrogen was found, however, in all fractions, being present in high concentration in the proteins and in the amide nitrogen and water-soluble substances precipitated by the Neuberg reagent from extracts of the leaves and stalks. As in the tobacco plant experiment, it was present in only low concentration in the filtrates from the Neuberg reagent precipitates. The effects on the stalk constituents were strikingly more intense than were those on the leaf constituents, though the actual quantities of t he several nitrogenous substances in t,he stalks were much smaller than in the leaves. SUMMARY When ammonium chloride that contains nit,rogen of atomic weight 15 is administered for a short period to rapidly growing plants, the isotope can be promptly detected in all parts of the tissues. The ammonia absorbed is rapidly assimilated into the nitrogen of amides and amino acids and into the proteins. The concentration of isotope in the nitrogen suggests greatest intensity of assimilation in the roots and least in the leaves; on the other hand the greatest quantity of isotopic nitrogen was found in the leaves. Much of the chemical change can be accounted for as a result of growth during the experimental period, but the quantity of isotope found in the proteins of the tissues was appreciably in excess of that to be expected from growth alone. This excess is apparently the result of continuous chemical interaction between the nitrogen of the tissue constituents and that of the nutrient. In the course of these reactions isotopic nitrogen is introduced into the proteins. This is held to furnish an example of the normal process of nitrogen assimilation. There is a close and striking analogy to the type of continuous chemical reaction in which the
Vickery, Pucher, Schoenheimer, Rittenberg 539 tissue proteins of mature animals in nitrogen equilibrium have already been observed to be concerned. BIBLIOGRAPHY 1. Rittenberg, D., Schoenheimer, R., and Keston, A. S., J. Biol. Chem., 128, 603 (1939). 2. Schoenheimer, R., Ratner, S., and Rittenberg, D., J. Biol. Chem., 130, 703 (1939). 3. Vickery, H. B., Pucher, G. W., Leavenworth, C. S., and Wakeman, A. J., Connecticut Agric. Exp. Stat., Bull. $74, 553 (1932). 4. Rittenberg, D., Keston, A. S., Rosebury, F., and Schoenheimer, R., J. Biol. Chem., 137, 291 (1939). 5. Vickery, H. B., J. Biol. Chem., 86, 657 (1925); Plant Physiol., 2, 303 (1927). 6. Keston, A. S., Rittenberg, D., and Schoenheimer, R., J. Biol. Chem., 137, 315 (1939). 7. Gregory, F. G., and Sen, G. K., Ann. Bot., 1, 521 (1937). See also Vickery, H. B., and Pucher, G. W., J. Biol. Chem., 128, 685 (1939); Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. S., Connecticut Agric. Exp. Stat., Bull. 424 (1939). 8. Vickery, H. B., Pucher, G. W., Schoenheimer, R., and Rittenberg, D., J. Biol. Chem., 129, 791 (1939).
THE ASSIMILATION OF AMMONIA NITROGEN BY THE TOBACCO PLANT: A PRELIMINARY STUDY WITH ISOTOPIC NITROGEN Hubert Bradford Vickery, George W. Pucher, Rudolf Schoenheimer and D. Rittenberg J. Biol. Chem. 1940, 135:531-539. Access the most updated version of this article at http://www.jbc.org/content/135/2/531.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/135/2/531.citation.full.h tml#ref-list-1