Nitrogen assimilation in soybean nodules

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Soil Science and Plant Nutrition ISSN: 0038-0768 (Print)

com/loi/tssp20 Nitrogen assimilation in soybean nodules

Ohyama & Kikuo Kumazawa (1980) Nitrogen assimilation in

1080/00380768.1980.10431204 Published online: 29 Mar 2012.

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1 Soil Science and Plant Nutrition ISSN: (Print) (Online) Journal homepage: Nitrogen assimilation in soybean nodules Takuji Ohyama & Kikuo Kumazawa To cite this article: Takuji Ohyama & Kikuo Kumazawa (1980) Nitrogen assimilation in soybean nodules, Soil Science and Plant Nutrition, 26:2, , DOI: / To link to this article: Published online: 29 Mar Submit your article to this journal Article views: 85 View related articles Citing articles: 13 View citing articles Full Terms & Conditions of access and use can be found at

Soil Sci. Plant Nutr., 26 (2), 205-213, 1980 NITROGEN ASSIMILATION IN SOYBEAN NODULES II.

2 Soil Sci. Plant Nutr., 26 (2), , 1980 NITROGEN ASSIMILATION IN SOYBEAN NODULES II. 15N 2 Assimilation in Bacteroid and Cytosol Fractions of Soybean Nodules Takuji OHYAMA and Kikuo KUMAZAWA Faculty 0/ Agriculture, The University 0/ Tokyo, Tokyo J 13, Japan Received August 14, 1979 UN assimilation was studied in bacteroid and cytosol fractions of soybean nodules. In the first experiment, after exposing the intact nodules to 16N a for 5 min and 10 min, most of the fixed UN was detected in cytosol fraction. In cytosol fraction, UN content of glutamine was the highest and followed by glutamic acid, alanine, and allantoin in this sequence, whereas, in bacteroid fraction, glutamic acid showed the highest UN content and alanine and glutamine followed. In the second experiment, 16N assimilation of various 16N-labeled compounds in the separated bacteroid and cytosol fractions was investigated. In the separated bacteroid fraction which was fed with 16NH" 16N was incorporated very rapidly into glutamic acid, alanine, and aspartic acid, but very slowly into glutamine. From these results, it was suggested that most of the fixed ammonia was exported to cytosol and assimilated via glutamine synthetase to glutamine, then via glutamate synthase to glutamic acid, and from these compounds various nitrogenous compounds were formed, but in bacteroids glutamate dehydrogenase and alanine dehydrogenase played an important role in the assimilation of fixed ammonia though quantitatively the contribution to ammonia assimilation in nodules was much less compared with cytosol. Additional Index Words: soybean nodule, nitrogen-ls, nitrogen assimilation, bacteroid and cytosol. In the nodule of leguminous plants, fixed nitrogen is metabolized in different ways between bacteroid and cytosol fractions (1, 15). In the bacteroid fraction of soybean, there was much amount of ammonia compared with other compounds, while in cytosol, allantoin and asparagine were present exclusively (15);- Many enzymatic studies have been done in separating bacteroid and cytosol fractions, regarding the early steps of nitrogen assimilation in nodules. High level of GS (glutamine synthetase) was consistently detected in cytosol fraction of various species of herbaceous legumes (4, 5, 9,11,18,19). and the activity ofgs was found to increase during nodule development over a time course which followed the induction of leghemoglobin and nitrogenase (19). Though the activity of GS has also been detected in bacteroid fraction (3, 5, 7, 10), its level was much lower than cytosol GS (5, 7, 9, 11, 18, 19) or GS of free living Rhizobium (19). GOGAT (glutamate synthase) have been reported in both the bacteroid (5, 10, 13, 18, 20, 21) and cytosol (4, 20) fractions 205

$206 T. OHYAMA and K. KUMAZAWA of various kinds of leguminous nodules. ROBERTSON and WARBURTON (20) reported that the level of GOGAT increased in cytosol fraction over the same time course as GS.$

3 206 T. OHYAMA and K. KUMAZAWA of various kinds of leguminous nodules. ROBERTSON and WARBURTON (20) reported that the level of GOGAT increased in cytosol fraction over the same time course as GS. GDH (glutamate dehydrogenase) activity was also detected both in bacteroid (10, 18) and cytosol (4, 18) fractions, and Km of GDH for ammonium was generally high compared with as for hydroxylamine (4) or ammonium (24). RYAN and FOTIRELL (21) showed that most of the total activities of GDH and Asp-AT (aspartate amino transferase) are located in cytosol, though Ala-AT (alanine amino transferase) and GOGAT are in bacteroid fraction of soybean nodules. But DUKE and HAM (6) reported that more than 90% of GDH activity is in bacteroid plus mitochondria fraction, and GRIMES and FOTIRELL (8) detected 98% Ala-AT in cytosol fraction. On the other hand, only a few isotope tracer studies have been reported on nitrogen assimilation of bacteroid and cytosol fractions distinctively. BERGERSEN (2) observed that after the exposure to UNa for 1 min more than 90% of the fixed nitrogen in the soluble fraction was detected as ammonia. And O'GARA and SHANMUGAN (14) exposed the free living cultures of Rhizobium japonicum to UN.. and showed that 15N was recovered as 15NH, from supernatant of culture medium. From the studies of UN pulse chase experiment (15) and the effects of metabolic inhibitors on the assimilation of UN-labeled nitrogenous compounds (17), it was concluded that the major portion of ammonia was initially assimilated via GS/GOGA T pathway. In this paper, UN assimilation was studied in bacteroid and cytosol fractions just after exposing the intact nodules to un 2, and also the metabolic pattern of various UN-labeled compounds in the separated bacteroid and cytosol fractions was studied. MATERIALS AND METHODS Plant. Soybean plants were inoculated with Rhizobium japonicum (NIAS, J-501) and grown in nitrogen-free solution as described before (17). Assimilation of un in bacteroid and cytosol fractions separated from intact nodules fed with UNa. The 44 days old plants cultivated in the glass-house were transferred in the phytotron (25 C, 10,000 lux), and the nodules attached to the intact roots were exposed to UNa (61.0 atom %, N2 : O 2 : He= 1 : 2 : 7) for 5 or 10 min using the UNafeeding apparatus as shown before (15). After exposure to UNa, the nodules were rapidly removed and macerated with a mortar and pestle in the chilled 0.2 M solbitol solution, and the macerate was filtered through 4 layers of nylon net (25 pm). The filtrate was centrifuged at 6,000 x a for 15 min, and separated into supernatant (cytosol) and precipitate (bacteroid) fractions. Each fraction was extracted with 80% ethanol, and 15N content of nitrogenous compounds were measured by optical emission spectrometry (15, 16). The amount of soluble nitrogen in each fraction was determined by isotopic dilution technique with UN-labeled alanine (30 atom %, 2 pg-n).

N Assimilation In Bacteroid and Cytosol of Nodule 207 15NH, assimilation. of bacteroids isolated from nodules.

4 N Assimilation In Bacteroid and Cytosol of Nodule NH, assimilation. of bacteroids isolated from nodules. The nodules attached to the roots of 47 days old p l a were n ~ s washed with ice-chilled water and detached from the roots. About 10 g nodules were macerated with a mortar and pestle in chilled T.B. (ph 7.5, 0.05 M Tris-HCI buffer containing 1 mm dithiothreitol, 0.2 M sorbitol, 0.2% insoluble polyvinylpyrrolidon, and 0.1% glucose), and the macerate was filtered and separated into bacteroid and cytosol fractions as described above. The bacteroid fraction was resuspended with T.B. and centrifuged again for complete separation from the cytosol fraction. Bacteroids were incubated at 25 C with 0.5 mm (15NH')2S0, (75 atom %) in 10 ml T.B. for I, 2, 5, 10, IS min and incubation was terminated and free nitrogenous compounds were separated with 80% ethanol. Their un abundance was determined. Assimilation of UN-labeled glutamine, glutamic acid, alanine, or glycine in bacteroid and cytosol fractions. Bacteroid and cytosol fractions were separated as described above, and incubated at 25 C for IS min with 15N-Iabeled 1 mm glutamine (ami doun, 49 atom %), 1 mm glutamic acid (50 atom %), 1 mm alanine (30 atom %), or 1 mm glycine (30 atom %) in T.B. The 15N abundance of 80% ethanol-extractable ami des and amino acids was determined, and 15N specific activity (16) was expressed as the percentage of nitrogen derived from labeled source. The. effect of metabolic inhibitors on nitrogen assimilation in isolated bacteroid fraction.,isolated bacteroid fraction was preincubated at 25 C for IS min with 10 mm m e t h i sulfoximine o n i n ~ (MSX) in 10 ml T.B., and 0.5 mm (15NH,)aSO, (75 atom %) was added and fed for IS min. Ten mm azaserine (AS) pretreatment and 1 mm glutamine (amido- 15 N, 49 atom %) incubation was carried out in the same manner. un_ incorporation into free nitrogenous compounds was measured. RESULTS AND DISCUSSION Table 1 shows the abundance and distribution of 15N in 80% ethanol-soluble nitrogen of bacteroid and cytosol fractions separated immediately after exposure of una gas to intact soybean nodules for 5 and 10 min. un abundance of both fractions at 10 min was approximately twice as high as those at 5 min, indicating that newly Table 1. The abundance and distribution of un in 80% EtOH-soluble fraction of bacteroid and cytosol separated from intact soybean nodules exposed to un,. un, exposure Atom % pg-n pg-fixed N % un time (min) excess per plant per plant distribution 5 Bacteroid Cytosol Bacteroid Cytosol lin, (61.0 atom %) : 0, : He-I: 2: 7.

5 208 T. OHYAMA and K. KUMAZAWA fixed nitrogen was incorporated steadily into these fractions. un abundance of cytosol fraction was remarkably higher than that of bacteroid in 5 and 10 min un, exposure. Distribution of fixed nitrogen in cytosol amounted to 97% and 96% of the total nitrogen in 80% ethanol-soluble fraction after 5 min and 10 min un, supply, respectively. These results indicate that most of the newly fixed nitrogen is transferred to cytosol soon after the fixation by nitrogenase in bacteroid. Figure I shows the incorporation of un into the principal nitrogenous compounds of bacteroid and cytosol fractions. It is obvious that the two fractions had quite different pattern of 15N incorporation, that is, in the bacteroid fraction, glutamic acid showed the highest un content, followed by alanine, and glutamine. On the other hand, in the cytosol fraction, the un content of glutamine was the highest, followed by glutamic acid, alanine, and allantoin plus allantoic acid in this sequence. The content of 15N in asparagine in both fractions increased very slowly. These results suggest that newly fixed ammonia may be assimilated to glutamic acid mainly via GDH in bacteroid fraction, and via GS/GOGAT pathway in cytosol fraction. Figure 2a shows the 15NH, assimilation of bacteroids isolated from soybean nodules. As in bacteroid, glutamic acid showed the highest amount, aspartic acid and alanine were almost the same, and glutamine was comparatively low (15), 15N was thought to be incorporated very rapidly into glutamic acid, alanine, and aspartic acid, but very slowly into glutamine. These results revealed that externally supplied ammonia was not assimilated by GS but possibly via GDH pathway to glutamic acid in 1:iacteroid. The shape of UN incorporation pattern of glutamic acid shows the hyperbolic curve and similar labeling pattern of aspartic acid follows, whereas alanine shows 1a 3 Ib., u '".,......!II!II., u.. ~ ~ e e os os < < Time (min) Time (min) Fig. 1. Incorporation of lin into nitrogenous compounds of bacteroid (Fig. 1a) and cytosol (Fig. 1b) fractions separated just after exposure of lin, to intact soybean nodules. o Glu, GIn, Ala, A Asn, 0 allantoin+allantoic acid.

N Assimilation in Bacteroid and Cytosol of Nodule 209 relatively steady un incorporation. These trends were also shown in the preliminary result (Fig.

6 N Assimilation in Bacteroid and Cytosol of Nodule 209 relatively steady un incorporation. These trends were also shown in the preliminary result (Fig. 2b) obtained from the experiment carried out in the previous year with the same experimental design. If alanine had been formed only by alanine transaminase accepting amino-group from glutamic acid, the 15N incorporation pattern should have followed glutamic acid like aspartic acid. However, the labeling pattern of alanine seems to be independent of glutamic acid. Therefore, in bacteroid fraction, ammonia may be assimilated directly to alanine by alanine dehydrogenase, which has high activity in bacteroid (7), rather than via GDH/Ala-AT pathway. As shown in Fig. 2a, nitrate (including nitrite) was formed from ammonia in isolated bacteroids. As previously reported un was incorporated into nitrate within a few minute of1 5 N 2 exposure to intact nodules especially in bacteroid fraction (I5). SIK et al. (23) reported that free living Rhizobium cultured with amino acids as the sole nitrogen source accumulated nitrite in medium, and suggested that Rhizobium could form nitrate. 15NH, added to cytosol fraction was neither assimilated to amides nor amino acids at all. The lack of GS activity in cytosol fraction might be due to the loss of energy source (ATP) for enzymatic reaction during the preparation. Tables 2 and 3 show UN content of nitrogenous compounds extracted from the isolated bacteroid and cytosol fractions which were fed with UN-labeled glutamine, glutamic acid, alanine, or glycine. As shown in Table 2, in bacteroid fraction, glutamine amido-n could be metabolized to glutamic acid, possibly by GOGAT which has been detected in bacteroids (5, 10, 18, 20, 21), and alanine showed relatively lower 15N content. Application of UN-labeled glutamic acid, alanine, and glycine suggested that aspartic acid and alanine were actively transaminated from glutamic acid, and that transamination between alanine and glycine could easily occur. 1.0, , 10 r , 2. 2b., III.... U ~ < ~ 0.5., III.. ~ Time (min) Time (min) Fig. 2. linh, assimilation of bacteroids isolated from soybean nodules. o Glu, GIn, Ala, A Asp,... nitratc+nitrite.

7 210 T. OHYAMA and K. KUMAZAWA In cytosol fraction (Table 3), glutamine was hardly metabolized to other nitrog-" enous compounds. The lack of GOGAT activity might be due to the loss or dilute concentration of reducing power (NAD(P)H) in cytosol fraction. Aspartic acid, serine, and alanine were actively transaminated from externatiy added glutamic acid. And amino-n of alanine could be transaminated to serine, glutamic acid, and aspartic acid, and that of glycine to alanine and other amino acids and amides. Table 4 shows the effect of MSX on 15NH, assimilation of isolated bacteroids and Table 5 shows the effect of AS on the glutamine amido- 15 N assimilation. Pretreatment with MSX which is a potent inhibitor of GS (12) did not repress UN incorporation into glutamic acid and alanine, but AS, an inhibitor of GOGAT (7, 12) Table 2. UN content of nitrogenous compounds in bacteroid fraction fed with UN-labeled glutamine (ami do-un), glutamic acid, alanine, or glycine. Separated N-compounds Glutamine UN-labeled compounds Glutamic acid Alanine Glycine Glutamine 22.2 N.D. Glutamate Aspartate Alanine Glycine N.D.- N.D. - N.D.: Not determined. un contents are expressed as UN specific activities: 100 x atom % excess of separated compound atom % excess of labeled compound N.D. 0.6 N.D N.D Table 3. UN content of nitrogenous compounds in cytosol fraction fed with UN-labeled glutamine (amido-n), glutamic acid, alanine, or glycine. Separated N-compounds UN-labeled compounds Glutamine Glutamic acid Alanine Glycine Glutamine Asparagine Glutamate Aspartate N.D Alanine Serine _ Glycine 0.00 N.D. N.D GABA N.D.: Not determined. lin contents are expressed as UN specific activities.

8 N Assimilation in Bacteroid and Cytosol of Nodule 211 depressed the un content of all amino acids. These reults support the previous consideration that in bacteroid, ammonia was assimilated by an alternative pathway such as GDH rather than GSjGOGAT system, though externally added glutamine could be metabolized to glutamic acid by GOGAT in bacteroid. Figure 3 shows the scheme on nitrogen flow in bacteroid and cytosol fractions of soybean nodules. Most of the fixed ammonia in bacteroid was exported to cytosol. Table 4. The effect of MSX on NH, assimilation of isolated soybean nodule bacteroids. Control +MSX Glutamine Glutamate Alanine UN atom % excess Table 5. The effect of AS on the glutamine amido-n assimilation of isolated soybean nodule bacteroids. Control +AS Glutamine Glutamate Aspartate Alanine UN atom % excess atom % UN-glutamine (amido label) was fed. (bacteroid) N2 (cytosol) [Nitrogenase]l 1/ ~ A D /[agkdh94] H I Ala ~ P y r NH _+-+NH, S. G 1 U, ~. [% Glu [GOGAT] Gly ' G l { [ ~ ~ / 1/ 1t\ ~ G l Y I.r Gl Asp Ala Asp ~ g t ~ ] ~ ser'" - -! [Transaminases] akg i [Transaminases] I lu [Asn synthetase] \ Asn " - Allantoin ~ Fig. 3. Scheme on nitrogen flow in bacteroid and cytosol fraction of soybean nodule.

9 212 T. OHYAMA and K. KUMAZA WA In cytosol, ammonia was immediately assimilated by GS to glutamine. Some parts of glutamine was metabolized by GOGAT producing glutamate, and the amino-nitrogen of glutamic acid could be transferred to keto acids to form various amino acids. Other parts of glutamine could be utilized to synthesize asparagine via glutaminedependent asparagine synthetase, which activity was shown in lupin nodule (22), and allantoin possibly via purine formation. Allantoin and asparagine were the major transport form of nitrogen. In bacteroid, fixed nitrogen may be firstly metabolized via GDH and ADH to form glutamic acid and alanine, and other amino acids can be synthesized by transamination. BROWN and DILWORTH (5) reported that in cultured Rhizobium under glucose limiting and with an excess of inorganic nitrogen, ammonia was assimilated via GDH, and neither GS nor GOGAT was detected in its extracts. We already reported that the proportion of ammonia in free nitrogenous compounds was exclusively high in bacteroids compared with cytosol. As suggested by Brown and Dilworth, it is possible that the high concentration of ammonia depressed bacteroid GS, and consequently most of the ammonia was excreted to cytosol, and GDH and ADH were dominant in ammonia assimilation in bacteroids. REFERENCES 1) ANTONIW, L.D. and SPRENT, J.I., Primary metabolites of Phaseolous vulgaris nodules, Phytochemistry, 17, (1978) 2) BERGERSEN, F.J., Ammonia-An early stable product of nitrogen fixation by soybean nodules, Aust. J. BioI. Sci., 18, 1-9 (1965) 3) BISHOP, P.E., GUEVARA, J.G., ENGELKE, J.A., and EVANS, H.J., Relation between glutamine synthetase and nitrogenase activities in the symbiotic association between Rhizobium Japonicum and Glycine max. Plant Physiol., 57, (1976) 4) BOLAND, M.J., FORDYCE, A.M., and GREENWOOD, R.M., Enzymes of nitrogen metabolism in legume nodules; a comparative studies, Aust. J. Plant Physiol., 5, (1978) 5) BROWN, C.M. and DILWORTH, M.J., Ammonia assimilation by Rhizobium cultures and bacteroids, J. Gen. Microbiol., 86, (1975) 6) DUKE, S.H. and HAM, E.G., The effect of nitrogen addition on N.-fixation and on glutamate dehydrogenase and glutamate synthase activities in nodules and roots of soybeans inoculated with various strains of Rhizobium Japonicum. Plant Cell Physiol., 17, (1976) 7) DUNN, S.D. and KLUCAS, R.V., Studies on possible routes of ammonium assimilation in soybean root nodule bacteroids, Can. J. Microbiol., 19, (1973) 8) GRIMES, H. and FOTTRELL, P.F., Enzymes involved in glutamate metabolism in legume root nodules, Nature, 212, (1966) 9) KLUCAS, R.V., Studies on soybean nodule senescence, Plant Physiol., 54, (1974) 10) KURZ, W.G.W., ROKOSH, D.A., and LARUE, T.A., Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroid, Can. J. Microbiol.,21, (1975) 11) MCPARLAND, R.H., GUEVARA, J.G., BECKER, R.R., and EVANS, H.J., The purification and properties of the glutamine synthetase from the cytosol of soya-bean root nodules, Biochem. J., 153, (1976) 12) MIFLIN, B.J., The pathway of nitrogen assimilation in plants. Phytochemistry, IS, (1976)

N Assimilation in Bacteroid and Cytosol of Nodule 213 lj) NAOATANI, H., SHIMIZU, M., and VALENTINE, R.C., The mechanism of ammonia assimilation in nitrogen fixing bacteria, Arch. Microbiol.

10 N Assimilation in Bacteroid and Cytosol of Nodule 213 lj) NAOATANI, H., SHIMIZU, M., and VALENTINE, R.C., The mechanism of ammonia assimilation in nitrogen fixing bacteria, Arch. Microbiol., 79, (1971) 14) O'GARA, F. and SHANMUOAN, K.T., Regulation of nitrogen fixation by Rhizobia. Export of fixed Na as NH" Biochim. Biophys. Acta, 437, (1976), 15) OHYAMA, T. and KUMAZAWA, K., Incorporation of l&n into various nitrogen compounds in intact soybean nodules after exposure to l&na gas, Soil Sci. Plant Nutr., 24, (1978) 16) OHYAMA, T. and KUMAZAWA, K., Assimilation and transport of nitrogenous compounds originated from UNa fixation and unoa absorption, Soil Sci. Plant Nutr., 25, 9-19 (1979) 17) OHYAMA, T. and KUMAZAWA, K., Nitrogen assimilation in soybean nodules. I. The role of GSI GOGATsystem in the assimilation of newly fixed ammonia, Soil Sci. Plant Nutr., 26, (1980) 18) PLANQuE, K., KENNEDY, I.R., DE VRIES, G.E., QurSPEL, A., and VAN BRUSSEL, A.A.N., Location of nitrogenase and ammonia assimilatory enzymes in bacteroids of Rhizobium leguminosarum and Rhizobium lupini, J. Gen. Microbiol., 102, (1974) 19) ROBERTSON, J.G., FARNDEN, K.J.F., WARBURTON, M.P., and BANKS, J.A.M., Induction of glutamine synthetase during nodule development in lupin, Aust. J. Plant Physiol., 2, (1975) 20) ROBERTSON, J.G., WARBURTON, M.P., and FARNDEN, K.J.F., Induction of glutamate synthase during nodule development in lupin, FEBS Lett., 55, (1975) 21) RYAN, E. and FOTTRELL, P.F., Subcellular localization of enzymes involved in the assimilation of ammonia by soybean root nodules, Phytochemistry, 13, (1974) 22) Scon, D.B. and FARNDEN, K.J.F., Ammonia assimilation in lupin nodules, Nature, 263, (1976) 23) Suc, T., KONDROSI, A., BARABAS, I., and SVAB, Z., Nitrate reductase and effectiveness in Rhizoblum, Proc.1st Int. Symp. Nitrogen Fixation, WSU Press, Washington, 2, (1976) 24) SLOOER, C., Assimilation of ammonia by glutamine synthetase and glutamate synthase in Na fixing bacterias from soybean nodules, Plant Physioi., 51, 34s (1974)

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12038 12042, September 1998 Plant Biology Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids JAMES K. WATERS, BOBBY L. HUGHES II, LARRY