School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, ACT 0200, Australia1
School of Life Sciences, University of Dundee, Dundee DD1 4HN, UK2
Biochemistry Department, The University of Western Australia, Nedlands, WA 6907, Australia3
Author for correspondence: Fraser J. Bergersen. Tel: +61 2 61254291. Fax: +61 2 61250313. e-mail: fraser.bergersen{at}anu.edu.au
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ABSTRACT |
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Keywords: alanine, alanine dehydrogenase, ammonia, nitrogen fixation, 15N balance
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INTRODUCTION |
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The basis for this interpretation was substantially due to the following experimental observations. (1) NH3 was the earliest 15N-labelled product of N2 fixation when detached root nodules of soybean (Glycine max Merr.; Bergersen, 1965 ) or serradella (Ornithopus sativa L.; Kennedy, 1966a
, b
) were incubated for short periods of time in atmospheres containing 15N2. (2) With anaerobically prepared soybean nodule bacteroids (the symbiotic form of Bradyrhizobium japonicum) in microaerobic, shaken assays with 15N2 in the gas phase, the principal product was 15NH3 (Bergersen & Turner, 1967
). (3) Later, 15NH3 was the principal product of 15N2 fixation by soybean bacteroids in an open, flow-reaction system in which a well-stirred suspension of soybean bacteroids was perfused with solutions containing dissolved O2 and 15N2; no 15N-labelled amino acids were detected. The long-accepted view was challenged by Waters et al. (1998)
who found that alanine, not NH3, was the principal 15N-labelled product when N2-fixing soybean bacteroids, purified on anaerobic sucrose density gradients, were supplied with malate and shaken in a gas mixture containing 15N2 and 0·008 atm O2. These authors concluded also that the earlier results arose from the use of bacteroid preparations that were contaminated with cytosolic enzymes from the host tissue, which released NH3 from the primary product, alanine. Allaway et al. (2000)
subsequently showed that both [15N]alanine and 15NH3 were produced by bacteroids prepared from nodules of Pisum sp. on anaerobic Percoll density gradients, but that the proportion of these products was altered by the conditions applied. When conditions were optimized for N2 fixation, NH3 was the first and major product formed, but alanine was formed at high bacteroid densities when NH3 accumulated (Allaway et al., 2000
). These authors also used a mutant strain of Rhizobium leguminosarum, defective in alanine catabolism, to produce N2-fixing nodules on peas. Such plants fix N2, but grow more slowly than plants nodulated by the wild-type, although the basis for this was not determined. Bacteroids from these nodules produced only NH3 in shaken assays. It was concluded that alanine was a secondary product of N2-fixing bacteroids; alanine amino-nitrogen arose from NH3, derived from N2 fixation, which accumulated in the experimental system used. More recently, Atkins & Thumfort (2001)
have reported that assimilation of 15N2 by nodulated roots of cowpea produced 15N-labelled products which showed no evidence that alanine was a precursor of the amide groups of glutamine or of the purine ring of ureides (the major N2-fixation product translocated from the nodules).
In the present paper we report experiments with density-gradient-purified soybean bacteroids, which were undertaken to verify the results of Waters et al. (1998) . Verification was not achieved. Instead, we found that NH3 was the sole significant 15N-labelled product of 15N2 fixation accumulated during 30 min in shaken assays with 0·0080·01 atm O2. Alanine, although sometimes found in low concentrations in flow chamber reactions, was not labelled with 15N in shaken, closed-system experiments. We conclude also that these and earlier results (Bergersen & Turner, 1967
, 1990
) were not due to contamination with host cytosolic enzymes as suggested by Waters et al. (1998)
and that NH3 is the principal product of N2 fixation in soybean.
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METHODS |
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Nodules and bacteroid suspensions.
Nodules aged about 35 days were picked from roots of soybean (Glycine max Merr. cv. Stevens, and sometimes for comparison, cv. Williams) inoculated at planting with B. japonicum strain USDA110c and grown in pots of sand, supplied twice weekly with McKnights nutrient solution free of combined nitrogen (Gibson, 1980 ). The nodules were washed in tap water, drained and blotted dry with paper towels and used immediately for preparation of bacteroid suspensions: (i) by the standard method of anaerobic homogenization in phosphate buffer (0·1 M, pH 7·4) containing sucrose (0·2 M), Mg2+ (2 mM), granular PVP (Polyclar) and filtration through Mira-cloth in a closed system under flowing argon [bacteroids were obtained by differential centrifugation and washed as described by Bergersen & Turner (1990)
]; or (ii) by homogenization in the same solution under argon in an anaerobic glove box, followed by separation of bacteroids by anaerobic centrifugation on Percoll (Pharmacia) density gradients and washing, essentially as described by Udvardi et al. (1988
; cf. Allaway et al., 2000
). Finally, the bacteroids were dispersed in reaction solution and an aliquot was saved for determination of dry weight after centrifugation and washing with distilled water.
Experimental systems.
Experiments in closed systems were based on those described by Waters et al. (1998) and Allaway et al. (2000)
, but some of the buffer constituents and concentrations were different. N2 fixation experiments were run in conical flasks (100 ml) closed with Suba Seals. Reaction solutions (9·0 ml per flask) contained 50 mM MOPS/KOH buffer, pH 7·4, 2 mM DL-malate and 0·2 M sucrose. After inserting the Suba Seals, the flask contents were degassed under vacuum for 10 min with periodic agitation on a manifold fitted with hypodermic needle connections and an Hg manometer (Turner & Gibson, 1980
). The flasks were flushed twice with Ar and then filled with gas mixtures containing 0·0080·02 atm O2 and N2 to 1 atm, from a screw-piston reservoir (Turner & Gibson, 1980
). They were brought to the reaction temperature (26 °C) before injecting 1·0 ml bacteroid suspension containing 36 mg (dry wt) bacteroids and shaking at 100 or 150 r.p.m. in a rotary shaker. Reactions were terminated by removing the seals, admitting air to inactivate nitrogenase, immediately chilling on ice and then centrifuging at 0 °C and 10000 g to separate bacteroids from the reaction solution. Products of N2 fixation were measured (below) in the supernatant reaction solution.
Experiments in the flow chamber system (Bergersen & Turner, 1990 ) were performed as described by Li et al. (2001)
, but samples of effluent were analysed for NH3, alanine and other amino acids as described below.
Detection of alanine-degrading enzymes adhering to bacteroids.
The presence of enzymes able to deaminate alanine and which adhered to bacteroids was sought in bacteroids prepared by differential centrifugation or by density gradient purification. Dehydrogenases were detected by following the reduction of NAD+ spectrophotometrically at 340 nm in assays containing 100 mM K-CAPS buffer (pH 10·0), alanine (4 mM), NAD+ (3 mM) and 3 mg (dry wt) bacteroids. Also, any alanine-dependent production of was sought in similar aerobic (non-N2-fixing) assays devoid of exogenous NAD+.
Endogenous alanine dehydrogenase.
Alanine dehydrogenase activities were determined in cell-free extracts prepared by disruption of suspensions in a French press. Liquid cultures of various strains of rhizobia were harvested in mid-exponential phase by centrifugation and resuspended in breakage buffer. Bacteroids were prepared from soybean nodules as described above and resuspended in breakage medium (TES/NaOH buffer, pH 7·5, containing 50 mM KCl, 5 mM MgSO4 and 5 mM DTT). The homogenates were clarified by centrifugation at 35000 g at 5 °C for 1 h to yield crude soluble extracts. Alanine dehydrogenase activity was estimated at 25 °C according to Allaway et al. (2000) and Smith & Emerich (1993)
. The apparent Km values for pyruvate and
were determined.
15N experiments.
15N2 gas was prepared from (15NH4)2SO4 (57·8 atoms% 15N; Isotec, Miamisburg, OH, USA) by oxidation with alkaline hypobromite (Bergersen, 1980 ) and purified by storage over alkaline potassium permanganate solution (to remove oxides of nitrogen) and then over dilute H2SO4 (to remove any residual NH3) (Burris, 1976
). Contamination of this gas with O2 was measured electrometrically as described below and its concentration adjusted to the desired level by addition of Ar and air after transfer at measured pressure to the screw-piston reservoir (see above). Typically, reaction gas mixtures used contained 0·008 atm O2, 0·5 atm Ar and 0·49 atm N2 (54·6 atoms% 15N) [At the elevation of Canberra, 1 atm is 705715 mm Hg (9495·3 kPa).] For experiments in shaken flasks (see above), a blank, unsealed control flask, containing all reaction components except 15N2, was placed on ice at zero time and thereafter treated in the same manner as the reaction flasks. Reactions were terminated as above after shaking for 30 min. The flask contents were analysed for N2 fixation products (see below).
Chemicals and analytical methods.
Except where indicated, all chemicals were of analytical grade purchased from Sigma. NH3 in reaction solutions and in fractions after Kjeldahl digestion and recovery by steam distillation (Bergersen, 1980 ) or by diffusion at pH 11 onto filter paper strips (Whatman No. 1; 15x4 mm) soaked in 0·5 M H2SO4, was determined colorimetrically by the ChaneyMarbach method (Bergersen, 1980
). Alanine and other amino acids were determined by HPLC analysis after derivitization at the Nucleic Acid and Applied Protein Chemistry Unit, Department of Plant Science, Waite Campus, University of Adelaide, Australia, or at the Ecosystems Research Group, Department of Botany, University of Western Australia.
The concentration of O2 contaminating the 15N2 used in N2 fixation experiments was determined in a sample (20 ml) of the gas collected in a gas-tight syringe and flushed through the previously Ar-flushed gas space (volume 5 ml) above 1 ml of stirred distilled water in the chamber of a pre-calibrated O2 electrode (Rank Bros.). The chamber was sealed before electrode currents were recorded and the zero checked after measurements by addition of a few crystals of sodium dithionite to the water layer.
In the 15N experiments, samples (1·0 ml) of the reaction flask contents were taken for analysis of total nitrogen by Kjeldahl digestion and distillation (Bergersen, 1980 ). The rest of the reaction mixture was separated into the supernatant (soluble) and bacteroid fractions by centrifugation at 10000 g for 10 min. The small quantities of free NH3
in the soluble fraction, being insufficient for 15N analysis, were recovered by diffusion onto paper strips (below) and after drying were supplemented with carrier nitrogen (50 µg) as (NH4)2SO4 of accurately known natural nitrogen abundance. For these analyses, the atoms% 15N before addition of carrier nitrogen (x) was calculated as:
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in which a =µg NH3 nitrogen in the sample before adding carrier, b=µg carrier nitrogen added, c=atoms% 15N measured in the sample plus carrier and d=atoms% 15N of the carrier nitrogen.
Bacteroid total nitrogen, soluble total nitrogen and the total nitrogen of the residue of the soluble fraction after removing NH3 nitrogen, were recovered by Kjeldahl digestion and distillation. Thus the distribution of all 15N incorporated from 15N2 could be determined. NH3 in distillates and in samples of supernatants was recovered by diffusion for >24 h onto strips of filter paper soaked in 0·5 M H2SO4 (see above; Bergersen, 1980 ). The filter paper strips were dried in tin capsules and submitted to total nitrogen and 15N analysis using an ANCA SL stable isotope analytical system (Europa Scientific) in the CSIRO Division of Plant Industry, Canberra, Australia. In addition to these analyses, the presence of [15N]alanine in the soluble fraction was sought by purifying any amino acids present by using chromatography on Sephadex (Pharmacia) SP-25 (Redgwell, 1980
), lyophilizing eluates and submitting them to GC-MS analysis after derivatization with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (Allaway et al., 2000
). The amount of 15N incorporated into alanine was assessed from the ratios of the mass peaks at m/e 260 and 261 (the principal fragments of the derivative of alanine), as follows:
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in which Rs and Rc are, respectively, the ratios mass 261/mass 260 for the mass spectral peaks of the sample (s) and the control (c). The calculation assumes that, apart from nitrogen, the isotopic ratios for all elements present in the fragment are the same in sample and control.
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RESULTS AND DISCUSSION |
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In both experiments alanine was present in the soluble fraction, but the GC-MS analysis revealed no significant excess of 15N in flasks containing bacteroids exposed to 15N2 for 30 min, compared with control flasks (Table 2). Values of R for the standard (Sigma) alanine were lower than those for the control (no 15N2) alanine. This may have been due to different isotopic composition of any of the elements in the Sigma alanine, but the m/e signals were free of signals from other chemicals which, at very low concentration, may alter the m/e signals of control or enriched experimental alanine. Additionally, the data for pure alanine were obtained at slightly higher concentration than for control and experimental alanine. In all cases, alanine was the most significant compound derivitized. It should be noted that had the standard alanine (Sigma; as used by Waters et al., 1998
) been used as the control at natural 15N abundance, the alanine from the reactions would appear to have been enriched with 15N (0·88 atoms% excess).
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Flow chamber experiments
Waters et al. (1998) stressed the need to use gradient-purified bacteroids which earlier work had shown to be free of host cytosolic enzyme activity and that the critical pO2 for alanine production by density-gradient-purified bacteroids in their shaken, closed system was 0·008 atm. Therefore, we sought alanine production in gradient-purified bacteroids and in bacteroids prepared by differential centrifugation, in flow chamber reactions at low steady-state concentrations of dissolved O2 ([O2 free]). Examples from these experiments are presented in Table 3
. It has been shown that N2 fixation into NH3 by bacteroids in the flow chamber system depends on respiration rates (Bergersen, 1997
). Sucrose-density-gradient-purified bacteroids, as used by Waters et al. (1998)
, generally had lower rates of respiration and NH3 production, but yielded no more alanine than bacteroids prepared by other methods (data not presented). Percoll-gradient-purified bacteroids were more active metabolically than sucrose-gradient-purified bacteroids and produced alanine at up to 19% of rates of NH3 production (Table 3
), whilst bacteroids prepared by the differential centrifugation method, although producing more NH3 per unit of respiration (Table 3
), produced little alanine at rates unrelated to respiration. N2 fixation into NH3 by these bacteroids in shaken assays continued for 3040 min, declining gradually thereafter (Fig. 1
), but in the flow chamber, steady rates were sustained easily for several successive periods of >20 min. Allaway et al. (2000)
suggested that failure to detect significant alanine in flow chamber effluents (Bergersen & Turner, 1990
) could have been due to continuous removal of NH3 from the flow chamber, thus preventing NH3 reaching a concentration needed for significant alanine formation.
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Alanine dehydrogenase activity
An unpurified, soluble extract of the bacteroid preparation, as used in the 15N experiment (Table 1), contained endogenous alanine dehydrogenase activity [>500 nmol NADH min-1 (mg protein)-1 in the aminating direction], with an apparent Km of 0·9 and 7 mM for pyruvate and
, respectively (Table 4
). This activity was greater than the activity of extracts of the same strain grown in succinate medium. These results indicated clearly that the enzyme activity had not been lost in the symbiotic state or during the isolation of bacteroids, and the failure to detect substantial alanine excretion from N2-fixing bacteroids did not result from such loss.
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The need for density-gradient bacteroid preparation
Waters et al. (1998) reported that bacteroids prepared by a differential centrifugation method (as used by Bergersen & Turner, 1967
, 1990
) were contaminated with enzymes originating from the host cells of the nodules. These contaminants may have degraded alanine to produce NH3, thus preventing identification of alanine as a major product of N2 fixation. It seemed to us that Waters et al. (1998)
may not have used exactly the same procedure as the other workers and so we tested bacteroid preparations, as used in the present paper, for the presence of such contamination. In neither Percoll-gradient-purified bacteroids nor bacteroids prepared by our fractional centrifugation method was alanine-dependent reduction of NAD+ detected (data not shown). There was a slow production of NH3 (<5 nmol NH3 mg-1 h-1) from bacteroids prepared by fractional centrifugation, but this was not stimulated in the presence of alanine concentrations up to 10 mM. Therefore, contamination of bacteroids with enzymes degrading alanine was not the cause of the failure to detect alanine as a major product of N2 fixation in the present work (Tables 1
, 2
) or in previous work (Bergersen & Turner, 1967
, 1990
). The finding suggests that the density gradient methods, which take longer and result in bacteroids with impaired metabolic properties (Table 3
), may not be necessary.
Conclusion
We conclude that NH3 is the principal product of N2 fixation by bacteroids from soybean nodules under a range of different conditions ex planta, including those used by Waters et al. (1998) . The reason for this difference between the two laboratories could not be determined from our experiments, but the possibility remains that sucrose-density-gradient-purified bacteroids (Waters et al., 1998
), in closed shaken assays, differ from those purified on Percoll density gradients.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 December 2001;
revised 10 February 2002;
accepted 18 February 2002.