Planta
Planta (1984) 162:32%333
9 Springer-Verlag 1984
Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings C.A. Atkins, B.J. Shelp, P.J. Storer and J.S. Pate Department of Botany, University of Western Australia, Nedlands, WA 6009, Australia
Abstract. During early development (up to 18 d after sowing) of nodules of an "effective" cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3:Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N 2 (in 80 At:20 02, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N 2 fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO2 (0.1-0.2 mM) relieved host-plant nitrogen deficiency caused by the At: O2 treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar:O2-grown nodules occurred at 18-20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).
Key words: Ammonia - Nitrogen fixation - Nodule - Senescence (root nodules) - Ureide - Vigna.
Rhizobium-legume host symbiosis (for a recent review, see Sutton 1983). Once successful infection has resulted in the establishment of a pre-emergent nodule structure (for review, see Newcomb 1981) within the legume root, transformation into a functional nodule is extremely rapid and involves a complex set of morphological and biochemical changes, especially in relation to the transition of meristematic plant cells to infected cells. These events are usually associated with extremely rapid growth by the nodule and a dramatic increase in nitrogenase activity (Atkins et al. 1980b). Studies of a wide range of legume nodules have shown that concurrent with the above changes there is an equally rapid rise in the activity of enzymes associated with ammonia assimilation in the host cells and it has been supposed (Robertson et al. 1975a, b; see review by Atkins 1982) that induction of these enzymes results from the onset of ammonia excretion by the bacteroids. In this paper, we attempt to determine directly the extent to which the nitrogenous products of nitrogenase activity might influence the differentiation of infected plant tissues and nodule functioning, by examining patterns of enzymic activity in nodules of cowpea formed in the presence of N 2 or in an atmosphere of Ar: 0 2 in which ammonia formation by nitrogenase is prevented. In a companion paper (Atkins et al. 1984) the general effects of Ar:O 2 on cowpea seedling development have been described, and it has been shown how nodule growth and structure are influenced by this gaseous treatment in the presence or absence of NO 3. Materials and methods
Introduction
Many factors are likely to be involved in regulating the development and maturation of an effective
Plant material. Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings nodulated with Rhizobium strain CB756 were grown in N-free or in NO3--supplemented liquid culture with a rooting atmosphere of air or Ar:O2 (80:20, v/v) as described in Atkins et al. (1984).
328
Extraction of nodules. Nodules were homogenized in a chilled mortar and pestle with a breaking medium of 5 0 r a m N-[2-hydroxy-I ,l-bis-(hydroxymethyl)methyl]glycine (Tricine)NaOH (pH 7.5) containing 50 mM mercaptoethanol (and where purine synthesis was assayed, 25 mM KCI and I mg CI 2 in addition) and separated into soluble (mainly of plant-cell origin) and insoluble (mainly bacteroid) fractions as described in Atkins et al. (1984). Tbe insoluble component was resuspended in homogenizing buffer, disrupted by twice passing it through a chilled French press, and the supernatant collected following centrifugation (Atkins 1974). Both fractions were desalted by passage through Sephadex G-25 (Pharmacia, Uppsala, Sweden) equilibrated with homogenizing buffer. For assay of de-novo purine synthesis, the soluble fraction was not desalted but was adjusted to 40% (w/v) polyethylene glycol 4000 (BDH Chemicals, Kew East, Vic., Australia) and the precipitate collected and redissolved as described earlier (Atkins et al. 1982).
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules I
Xylem-sap collection and analysis. Bleeding exudate was collected for l-h periods from the cut stumps of at least five decapitated plants at each sampling time. The samples of sap from individual plants were pooled and, after recording the volume, were frozen and stored at - 1 5 ~ C prior to analysis for ureide content (Herridge et al. 1978).
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Enzyme assays. Enzymatic activity was measured at 30 ~ C. Hydroxybutyrate oxidoreductase (EC 1.1.1.30) was assayed in the extract of the insoluble fraction as described in Shelp eta]. (1983). Allantoinase (EC3.5.2.5), NAD-glutamate synthase (EC 2.6.1.53), glutamine synthetase (EC 6.3.1.2), urate oxidase (EC 1.7.3.3), xanthine oxidoreductase (EC 1.2.3.2), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and aspartate-ketoglutarate aminotransferase (EC 2.6.1.1) were assayed in the soluble extract as described in Atkins et al. (1980b). Assay of N A D glutamate oxidoreductase (EC 1.4.1.2) was by the method of Atkins et al. (1980b) except that Ca 2+ was added to a final concentration of 1 raM. De-novo purine synthesis was assayed as metabolism of [14C]-glycine to [~4C]inosine monophosphate (IMP) and [~C]aminoimidazol carboxamide ribonucleotide (AICAR) in the 40%-polyethyleneglycol-precipitated fraction of the soluble extract according to Atkins et al. (1982). Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin (Commonwealth Serum Laboratories, Melbourne, Vic., Australia) as standard, and leghaemoglobin as the pyridine haemochrome as described by Appleby (1969 b). Nitrogenase (EC 1.7.99.2) activity was assayed as the rate of H z evolution by nodulated root systems of plants during culture or as the rate of CzH z reduction by detached segments of nodu/ated roots, The H 2 content of samples of the gas streams leaving the enclosed root atmospheres of pots was measured using a gas liquid chromatograph (Shimadzu, Tokyo, Japan; GC-6AM) equipped with a 2-m column of molecular sieve 5A (100-200mesh; Waters Associates, Sydney, NSW, Australia) and a thermal conductivity detector. Gas samples were collected during a 1-h period in the middle of the photoperiod of each day of the experiment. For the C2H2-reduction assay, freshly harvested root segments were enclosed in 34-cm 3 serum vials with an atmosphere of air containing 10% (v/v) C2H 2. Both C2H 2 and C2H ~ content were measured by gas chromatography (Shimadzu GC-6AM chromatograph equipped with flame-ionization detectors and 2-m columns of 100120 mesh Porapak-N [Waters Associates] held at 100~ after 0, 15 and 30 min at 30 ~ C. Activity was calculated from the average of the rates for the two ~5-rain periods. Fresh weight of nodules enclosed in the assay vials was recorded after the assay was complete.
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14 18 22 days after sowing Fig. 1. Evolution of H 2 into the effluent gas stream from the root atmospheres of cowpea grown in water culture with the roots maintained in air or Ar:O2 (80:20; v/v). Values are means + SE (N= 3). The air or Ar:Oz gas streams were maintained from 4 to 24 d after sowing
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Results
Effect of N 2 deficiency on nitrogenase activity and leghaemoglobin content of nodules. Profiles of increasing rates of H 2 production (Fig. 1) by intact
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
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Fig. 4A, B. Effect of air or Ar:O 2 (80:20; v/v) around the nodulated root systems of water-cultured cowpea on A, the concentration of ureides in xylem sap, and B, the rate of exudation of ureide (i.e. concentration X volume of sap collected in I h). Values for the air and Ar:O 2 treatments are means • SE (N= 5), For treatments with NO3 the data are from a single experiment. Treatments were as outlined in the legend for Fig. 2
Effect of N 2 deficiency on enzymes of ammonia assimilation and ureide synthesis in nodules. X y l e m sap collected f r o m plants grown in air showed high levels o f ureide, increasing f r o m a r o u n d 4 m M at 14 d to almost 10 m M at 24 d (Fig. 4A). T h e conc e n t r a t i o n in sap f r o m A r : O z - g r o w n plants was consistently m u c h lower, remaining at a b o u t 2 m M t h r o u g h o u t the experiment (Fig. 4A). A d d i t i o n o f N O ~ led to an overall reduction in the concentration o f ureides in xylem sap in b o t h the A r : O z and air treatments (Fig. 4A).
330
C.A. A t k i n s et al. : N n u t r i t i o n a n d N fixation in '
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The rate at which xylem sap exuded from the plants varied markedly between the treatments, mean values ( _ standard error of the mean; n = 12) over the 14-24-d period being 12.9-t-2.3 gl h - ~ for the air-grown and 3.8___0.4 gl h -1 for the A r : O zgrown plants. Addition of NO~- greatly stimulated the exudation rate and the comparable mean values (n=8) were 1 0 0 + 1 2 g l h -1 plant -1 for air+NO;- plants and 95+_6 for A r : O 2 + N O ~ plants. Amounts of ureide in sap on an hourly basis were calculated by compounding the sap concentration values of Fig. 4A with exudation rates at each collection time. The data (Fig. 4B) indicated greatest export of ureide in the air-grown plants supplemented with NO3-, extremely low export from Ar:Oz-grown plants, and intermediate levels of export in the air and Ar:O 2 treatments. In air, in the presence or absence of added NO~-, export of ureide increased over the time of the experiment, while in Ar: 0 2, export remained very low (Ar:O2-NO~-) or declined progressively (ar'O2 + N O ; ) (Fig. 4B). A wide range of enzymes, involved in assimilation of ammonia, and in synthesis of purines and of ureides were assayed in extracts of nodules from plants grown in air, in Ar:O 2 or in Ar:O 2 with addition of NO;-. The results shown in Fig. 5 are from a single experiment in the case of air- and Ar:O2-grown nodules, and a separate experiment for those grown in Ar:O2 and supplemented with
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The levels of activity of enzymes of ammonia assimilation (glutamine synthetase, glutamate synthase, aspartate aminotransferase) and ureide biosynthesis (de-novo purine synthesis, xanthine oxidoreductase, urate oxidase, allantoinase) were generally greater in air-grown than in Ar:O2-grown nodules (Fig. 5). This was also the case for phosphoenolpyruvate carboxylase while for glutamate oxidoreductase there were no clear differences between the treatments. For hydroxybutyrate oxidoreductase the highest levels of enzyme activity were found in Ar:O2-grown nodules (Fig. 5). The most marked effect of the Ar" 0 2 treatment was on enzymes of ureide biosynthesis. The level of de-novo purine synthesis was negligible in extracts of Ar" O2-grown nodules and although measurable activities of xanthine oxidoreductase and urate oxidase were detected in these nodules, the presence of air greatly increased the levels of both enzymes (Fig. 5). In some cases the pattern of enzyme activity with time was altered by addition of NO;-, but in general, this supplementation of N supply did not substantially increase the level of enzymes in Ar: O2-grown nodules (Fig. 5).
C.A. A t k i n s et al. : N n u t r i t i o n a n d N fixation in
Vigna n o d u l e s
Discussion
Effect of N 2 deficiency on pathways of ammonia assimilation. In a previous study (Atkins et al. J 984) it was demonstrated that early development (up to 18 d after sowing) of cowpea nodules was very similar in air and in Ar: 0 2. Rates of growth and structural development were comparable and, provided that the Ar:O2-treated plants were supplemented with N O j , their non-fixing nodules achieved protein contents comparable to those of air-grown nodules. Subsequently, however, the course of development in Ar: 0 2 diverged markedly from that of normal nodule development in air, with the Ar:O2-treated nodules exhibiting premature senescence at 20 d, and even when plant N deficiency had been relieved by added NO 3, senescence under At: 02 was delayed by only a few days. When developing in air, the functioning symbiosis matured progressively with increasing levels of export of reduced N to the host up to 24 d after sowing (Fig. 4). While reducing markedly the export of N in xylem, culture of nodules in Ar:O 2 did not reduce significantly nitrogenase activity or Ieghaemoglobin development during the first ~8 d after sowing. Thus, absence of N 2 failed to alter the derepression of those plant and bacteroid-associated biochemical systems involed in the establishment of nitrogenase functioning in the developing nodule. In this respect the "effective" and "inoperative" (Ar:Oz-grown) nodules were highly comparable. To a greater or lesser extent all enzymes likely to be associated with the assimilation of ammonia and the synthesis of ureides (Fig. 5) were markedly stimulated by N2, and although the effector may be different in each case, the response was indicative of a regulatory role for either ammonia or products of its assimilation. Not all enzymes of N metabolism showed clear stimulation; aspartate aminotransferase and allantoinase were less sensitive while glutamate oxidoreductase was unaffected (Fig. 5). Furthermore, phosphoenolpyruvate carboxylase, an enzyme which may have a more general anaplerotic role in the carbon metabolism of nodules (Christeller et al. 1977; Cookson et al. 1980), showed a marked stimulation by N 2 (Fig. 5), perhaps indicating that other metabolic functions of the symbiosis were also regulated by the ammonia supply. Except for the enzymes of de-novo purine synthesis, all enzymes of N metabolism assayed were readily detected, albeit at reduced levels, in Ar:O 2grown nodules (Fig. 5). This was not surprising for enzymes such as glutamine synthetase, gluta-
331
mate synthase and aspartate aminotransferase, which catalyse reactions common to most plant tissues (see review by Miflin and Lea 1980), but for the enzymes of purine oxidation, xanthine oxidoreductase and urate oxidase, activities in Ar: 0 2grown nodules were considerably higher than those normally detected in other tissues of cowpea (Herridge et al. 1978) or in tissues and isolated cells of non-leguminous plants (Barankiewicz and Paszkowski 1980; Nguyen and Nato 1983). Although substantial transfer of ammonia from the bacteroid may have resulted in stimulation of the pathways of N metabolism, the high level of transfer associated with an "effective" symbiosis was apparently not necessary for derepression of these enzymes. The response of de-novo purine synthesis to N 2 deficiency was exceptional in that virtually no activity was detected in nodules grown in Ar:O 2 whether or not NO~- was added (Fig. 5). The pathway, measured in tissue extracts by synthesis of labeled inosine monophosphate from [14C]glycine, involves activity of at least eleven separate enzymes (see review by Atkins 1982) and it is conceivable that in Ar:O 2 repression or deletion of just one of these could have prevented activity. Labeled intermediates of the de-novo purine sequence, such as aminoimidazole carboxamide ribonucleotide or formyl-glycinamide ribonucleotide, which might have been expected to accumulate if only a part of the sequence was deleted (Atkins et al. 1982), were however not detected. In view of the fact that the whole metabolic pathway of inosine monophosphate synthesis from phosphoribosylpyrophosphate has been shown to be localized within the plastids of nodule cells (Shelp et al. 1983), it seems likely that the total sequence remained repressed in the absence of a pronounced flux of ammonia from the bacteroids. Interestingly, in other tissues of cowpea, assimilation of ammonia resulting from NO~ reduction does not lead to substantial ureide synthesis but rather to formation of amides and amino acids (Atkins et al. 1980a) as is the case in nodules of "non-ureideproducing" legumes (Atkins 1982). Induction of the ureide pathway in nodules, especially of the plastid-located de-novo purine sequence, may be regarded as a specific feature, like the formation of leghaemoglobin and other special plant proteins (nodulins; Legocki and Verma 1979), of an effective symbiosis. Effect of N 2 deficiency on bacteroid enzymes. The consequent N deficiency associated with AriO 2 treatment resulted in a marked reduction of bacte-
332
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
roid-protein levels even at 14 d after sowing (Atkins et al. 1984), and although addition of NO~ overcame N deficiency and greatly stimulated protein levels in nodules, most of the " e x t r a " protein was formed in the plant cells, with little transfer of N to the bacteroids (Atkins et al. 1984). Despite this N deficiency, nodules from Ar:O2-grown plants showed nitrogenase levels comparable with those in air up to 16 d after sowing and, even after the onset of senescence, marked activity was retained up to 24 d after sowing. In fact, the specific activity of C2H 2 reduction, expressed on a bacteroid-protein basis (calculated from data of Fig. 2 and Atkins etal. 1984), was similar in Ar:Og-grown (5.1 _+0.7 gmol h - 1 rag- 1) and air-grown nodules (4.4_+ 1.9) throughout the experiment. Hydroxybutyrate oxidoreductase activity was markedly stimulated in A r : O z (Fig. 5). Interestingly, increased levels of this bacteroid enzyme have been noted previously in normally senescing nodules of soybean (Stripf and Werner 1978) or following treatments which induced premature nodule senescence (Wong and Evans 1971). Earlier studies using Rhizobium strain CB756 in a number of symbioses with cowpea (Rainbird et al. 1983; Schubert et al. 1978), have inferred the presence of a unidirectional uptake hydrogenase. The very sharp decline in H 2 evolution in both nodule types by 15 or 16 d (Fig. 1), despite continued high rates of C2H 2 reduction, probably reflects the sudden activity of such an enzyme in H 2 uptake. Continued H 2 release in Ar:O2 while the rates declined to zero in air (Fig. 1) indicates, however, that there was considerably less effective recycling of H 2 in A r : O 2 compared with air. Apparently the uptake hydrogenase, though initially induced, was not maintained in the absence of N 2, Thus, although in Ar: 0 2 there was continued utilization of translocated sugar in respiratory metabolism, the efficiency of carbon use by nodules was apparently reduced. In this context it will be interesting to see if the special pathways of electron transport and oxidative phosphorylation, formed in bacteroids under conditions of low 0 2 tension and specifically associated with efficient nitrogenase functioning (Appleby 1969a), are also derepressed at very low levels of N 2.
References
The authors wish to thank P. Sanford for skilled technical assistance. The work was supported by grants from the Australian Research Grants Scheme (to C.A.A.), the Wheat Industry Research Council of Australia (to C.A.A. and J.S.P.) and by a National Science and Engineering Research Council of Canada post-doctoral fellowship (to B.J.S.).
Appleby, C.A. (1969 a) Electron transport systems of Rhizobium japonicum: haemoprotein P-450, other CO-reactive pigments, cytochromes and oxidases in bacteroids from N z fixing root nodules. Biochem. Biophys. Acta 172, 71-87 Appleby, C.A. (1969b) Properties of leghaemoglobin in vivo and its isolation as ferrous oxyleghaemoglobin. Biochim. Biophys. Acta 188, 222-229 Atkins, C.A. (1974) Occurrence and some properties of carbonic anhydrases from legume root nodules. Phytochemistry 13, 93-98 Atkins, C.A. (1982) Ureide metabolism and the significance of ureides in legumes. In: Advances in agricultural microbiology, pp. 211-242, Subba Rao, N.S., ed. Oxford and IBH Publishing Co., New Delhi, India Atkins, C.A., Pate, J.S., Griffiths, G.J., White, S.T. (1980a) Economy of carbon and nitrogen in nodulated and nonnodulated (NO3-grown) cowpea (Vigna unguiculata (L.) Walp.). Plant Physiol. 66, 978-983 Atkins, C.A., Rainbird, R.M., Pate, J.S. (1980b) Evidence for a purine pathway of ureide synthesis in N2-fixing nodules of cowpea (Vigna unguiculata (L.) Walp). Z. Pflanzenphysiol. 97, 249-260 Atkins, C.A., Ritchie, A., Rowe, P.B., McCairns, E., Sauer, D. (1982) De novo purine synthesis in nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp) and soybean (Glycine max L. Merr). Plant Physiol. 70, 55-60 Atkins, C.A., Shelp, B.J., Kuo, J., Peoples, M.B., Pate, J.S. (1984) Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings. Planta 162, 316-326 Barankiewicz, J., Paszkowski, J. (1980) Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves. Biochem. J. 186, 343-350 Christeller, J.T., Laing, W.A., Sutton, W.D. (1977) Carbon dioxide fixation by lupin root nodules. I. Characterisation, association with phosphoenolpyruvate carboxylase, and correlation with nitrogen fixation during nodule development. Plant Physiol. 60, 47-50 Cookson, C., Hughes, H., Coombs, J. (1980) Effects of combined nitrogen on anapleurotic carbon assimilation and bleeding sap composition in PhaseoIus vulgar& L. Planta 148, 429-433 Herridge, D.F., Atkins, C.A., Pate, J.S., Rainbird, R.M. (1978) Allantoin and allantoic acid in the nitrogen economy of the cowpea (Vigna unguiculata (L.) Walp). Plant Physiol. 62, 495-498 Legocki, R.P., Verma, D.P.S. (1979) A nodule-specific plant protein (nodulin-35) from soybean. Science 205, 190-193 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 Miflin, B.J., Lea, P.J. (1980) Ammonia assimilation. In: The biochemistry of plants, vol. 5 : Amino acids and derivatives, pp. 169-202, Miflin, B.J., ed. Academic Press, New York London Newcomb, W. (1981) Nodule morphogenesis and differentiation. Int. Rev. Cytol., Suppl. 13, 247-298 Nguyen, J., Nato, A. (1983) In vitro study of the xanthine dehydrogenase from illuminated or darkened leaves. Physiol. Plant. 59, 73-78 Rainbird, R.M., Atkins, C.A., Pate, J.S., Sanford, P. (1983) Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea. Plant Physiol. 71, 122-127 Robertson, J.G., Farnden, K.J.F., Warburton, M.P., Banks, J.A.M. (1975a) Induction of glutamine synthetase during
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
333
nodule development in lupin. Aust. J. Plant Physiol. 2, 265-272 Robertson, J.G., Warburton, M.P., Farnden, K.J.F. (1975b) Induction of glutamate synthase during nodule development in lupin. FEBS Lett. 55, 33-37 Schubert, K.R., Jennings, N.T., Evans, H.J. (1978) Hydrogen reactions of nodulated leguminous plants. II. Effects on dry matter accumulation and nitrogen fixation. Plant Physiol. 61,398-401 Shelp, B.J., Atkins, C.A., Storer, P.J., Canvin, D.T. (1983) Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata (L.) Walp.). Arch. Biochem. Biophys. 224, 429-441
Stripf, R., Werner, D. (1978) Differentiation of Rhizobiumjaponicum. II. Enzymatic activities in bacteroids and plant cytoplasm during the development of nodules of Glycine max. Z. Naturforsch. Teil C 33, 373-381 Sutton, W.D. (1983) Nodule development and senescence. In: Nitrogen fixation, vol. 3: Legumes, pp. 144-212, Broughton, W.J., ed. Clarendon Press, Oxford, UK Wong, P.P., Evans, H.J. (1971) Polyhydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiol. 47, 750-755 Received 2 March; accepted 7 May 1984
Planta (1984) 162:32%333
9 Springer-Verlag 1984
Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings C.A. Atkins, B.J. Shelp, P.J. Storer and J.S. Pate Department of Botany, University of Western Australia, Nedlands, WA 6009, Australia
Abstract. During early development (up to 18 d after sowing) of nodules of an "effective" cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3:Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N 2 (in 80 At:20 02, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N 2 fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO2 (0.1-0.2 mM) relieved host-plant nitrogen deficiency caused by the At: O2 treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar:O2-grown nodules occurred at 18-20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).
Key words: Ammonia - Nitrogen fixation - Nodule - Senescence (root nodules) - Ureide - Vigna.
Rhizobium-legume host symbiosis (for a recent review, see Sutton 1983). Once successful infection has resulted in the establishment of a pre-emergent nodule structure (for review, see Newcomb 1981) within the legume root, transformation into a functional nodule is extremely rapid and involves a complex set of morphological and biochemical changes, especially in relation to the transition of meristematic plant cells to infected cells. These events are usually associated with extremely rapid growth by the nodule and a dramatic increase in nitrogenase activity (Atkins et al. 1980b). Studies of a wide range of legume nodules have shown that concurrent with the above changes there is an equally rapid rise in the activity of enzymes associated with ammonia assimilation in the host cells and it has been supposed (Robertson et al. 1975a, b; see review by Atkins 1982) that induction of these enzymes results from the onset of ammonia excretion by the bacteroids. In this paper, we attempt to determine directly the extent to which the nitrogenous products of nitrogenase activity might influence the differentiation of infected plant tissues and nodule functioning, by examining patterns of enzymic activity in nodules of cowpea formed in the presence of N 2 or in an atmosphere of Ar: 0 2 in which ammonia formation by nitrogenase is prevented. In a companion paper (Atkins et al. 1984) the general effects of Ar:O 2 on cowpea seedling development have been described, and it has been shown how nodule growth and structure are influenced by this gaseous treatment in the presence or absence of NO 3. Materials and methods
Introduction
Many factors are likely to be involved in regulating the development and maturation of an effective
Plant material. Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings nodulated with Rhizobium strain CB756 were grown in N-free or in NO3--supplemented liquid culture with a rooting atmosphere of air or Ar:O2 (80:20, v/v) as described in Atkins et al. (1984).
328
Extraction of nodules. Nodules were homogenized in a chilled mortar and pestle with a breaking medium of 5 0 r a m N-[2-hydroxy-I ,l-bis-(hydroxymethyl)methyl]glycine (Tricine)NaOH (pH 7.5) containing 50 mM mercaptoethanol (and where purine synthesis was assayed, 25 mM KCI and I mg CI 2 in addition) and separated into soluble (mainly of plant-cell origin) and insoluble (mainly bacteroid) fractions as described in Atkins et al. (1984). Tbe insoluble component was resuspended in homogenizing buffer, disrupted by twice passing it through a chilled French press, and the supernatant collected following centrifugation (Atkins 1974). Both fractions were desalted by passage through Sephadex G-25 (Pharmacia, Uppsala, Sweden) equilibrated with homogenizing buffer. For assay of de-novo purine synthesis, the soluble fraction was not desalted but was adjusted to 40% (w/v) polyethylene glycol 4000 (BDH Chemicals, Kew East, Vic., Australia) and the precipitate collected and redissolved as described earlier (Atkins et al. 1982).
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules I
Xylem-sap collection and analysis. Bleeding exudate was collected for l-h periods from the cut stumps of at least five decapitated plants at each sampling time. The samples of sap from individual plants were pooled and, after recording the volume, were frozen and stored at - 1 5 ~ C prior to analysis for ureide content (Herridge et al. 1978).
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Enzyme assays. Enzymatic activity was measured at 30 ~ C. Hydroxybutyrate oxidoreductase (EC 1.1.1.30) was assayed in the extract of the insoluble fraction as described in Shelp eta]. (1983). Allantoinase (EC3.5.2.5), NAD-glutamate synthase (EC 2.6.1.53), glutamine synthetase (EC 6.3.1.2), urate oxidase (EC 1.7.3.3), xanthine oxidoreductase (EC 1.2.3.2), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and aspartate-ketoglutarate aminotransferase (EC 2.6.1.1) were assayed in the soluble extract as described in Atkins et al. (1980b). Assay of N A D glutamate oxidoreductase (EC 1.4.1.2) was by the method of Atkins et al. (1980b) except that Ca 2+ was added to a final concentration of 1 raM. De-novo purine synthesis was assayed as metabolism of [14C]-glycine to [~4C]inosine monophosphate (IMP) and [~C]aminoimidazol carboxamide ribonucleotide (AICAR) in the 40%-polyethyleneglycol-precipitated fraction of the soluble extract according to Atkins et al. (1982). Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin (Commonwealth Serum Laboratories, Melbourne, Vic., Australia) as standard, and leghaemoglobin as the pyridine haemochrome as described by Appleby (1969 b). Nitrogenase (EC 1.7.99.2) activity was assayed as the rate of H z evolution by nodulated root systems of plants during culture or as the rate of CzH z reduction by detached segments of nodu/ated roots, The H 2 content of samples of the gas streams leaving the enclosed root atmospheres of pots was measured using a gas liquid chromatograph (Shimadzu, Tokyo, Japan; GC-6AM) equipped with a 2-m column of molecular sieve 5A (100-200mesh; Waters Associates, Sydney, NSW, Australia) and a thermal conductivity detector. Gas samples were collected during a 1-h period in the middle of the photoperiod of each day of the experiment. For the C2H2-reduction assay, freshly harvested root segments were enclosed in 34-cm 3 serum vials with an atmosphere of air containing 10% (v/v) C2H 2. Both C2H 2 and C2H ~ content were measured by gas chromatography (Shimadzu GC-6AM chromatograph equipped with flame-ionization detectors and 2-m columns of 100120 mesh Porapak-N [Waters Associates] held at 100~ after 0, 15 and 30 min at 30 ~ C. Activity was calculated from the average of the rates for the two ~5-rain periods. Fresh weight of nodules enclosed in the assay vials was recorded after the assay was complete.
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14 18 22 days after sowing Fig. 1. Evolution of H 2 into the effluent gas stream from the root atmospheres of cowpea grown in water culture with the roots maintained in air or Ar:O2 (80:20; v/v). Values are means + SE (N= 3). The air or Ar:Oz gas streams were maintained from 4 to 24 d after sowing
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Results
Effect of N 2 deficiency on nitrogenase activity and leghaemoglobin content of nodules. Profiles of increasing rates of H 2 production (Fig. 1) by intact
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
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Fig. 4A, B. Effect of air or Ar:O 2 (80:20; v/v) around the nodulated root systems of water-cultured cowpea on A, the concentration of ureides in xylem sap, and B, the rate of exudation of ureide (i.e. concentration X volume of sap collected in I h). Values for the air and Ar:O 2 treatments are means • SE (N= 5), For treatments with NO3 the data are from a single experiment. Treatments were as outlined in the legend for Fig. 2
Effect of N 2 deficiency on enzymes of ammonia assimilation and ureide synthesis in nodules. X y l e m sap collected f r o m plants grown in air showed high levels o f ureide, increasing f r o m a r o u n d 4 m M at 14 d to almost 10 m M at 24 d (Fig. 4A). T h e conc e n t r a t i o n in sap f r o m A r : O z - g r o w n plants was consistently m u c h lower, remaining at a b o u t 2 m M t h r o u g h o u t the experiment (Fig. 4A). A d d i t i o n o f N O ~ led to an overall reduction in the concentration o f ureides in xylem sap in b o t h the A r : O z and air treatments (Fig. 4A).
330
C.A. A t k i n s et al. : N n u t r i t i o n a n d N fixation in '
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The rate at which xylem sap exuded from the plants varied markedly between the treatments, mean values ( _ standard error of the mean; n = 12) over the 14-24-d period being 12.9-t-2.3 gl h - ~ for the air-grown and 3.8___0.4 gl h -1 for the A r : O zgrown plants. Addition of NO~- greatly stimulated the exudation rate and the comparable mean values (n=8) were 1 0 0 + 1 2 g l h -1 plant -1 for air+NO;- plants and 95+_6 for A r : O 2 + N O ~ plants. Amounts of ureide in sap on an hourly basis were calculated by compounding the sap concentration values of Fig. 4A with exudation rates at each collection time. The data (Fig. 4B) indicated greatest export of ureide in the air-grown plants supplemented with NO3-, extremely low export from Ar:Oz-grown plants, and intermediate levels of export in the air and Ar:O 2 treatments. In air, in the presence or absence of added NO~-, export of ureide increased over the time of the experiment, while in Ar: 0 2, export remained very low (Ar:O2-NO~-) or declined progressively (ar'O2 + N O ; ) (Fig. 4B). A wide range of enzymes, involved in assimilation of ammonia, and in synthesis of purines and of ureides were assayed in extracts of nodules from plants grown in air, in Ar:O 2 or in Ar:O 2 with addition of NO;-. The results shown in Fig. 5 are from a single experiment in the case of air- and Ar:O2-grown nodules, and a separate experiment for those grown in Ar:O2 and supplemented with
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The levels of activity of enzymes of ammonia assimilation (glutamine synthetase, glutamate synthase, aspartate aminotransferase) and ureide biosynthesis (de-novo purine synthesis, xanthine oxidoreductase, urate oxidase, allantoinase) were generally greater in air-grown than in Ar:O2-grown nodules (Fig. 5). This was also the case for phosphoenolpyruvate carboxylase while for glutamate oxidoreductase there were no clear differences between the treatments. For hydroxybutyrate oxidoreductase the highest levels of enzyme activity were found in Ar:O2-grown nodules (Fig. 5). The most marked effect of the Ar" 0 2 treatment was on enzymes of ureide biosynthesis. The level of de-novo purine synthesis was negligible in extracts of Ar" O2-grown nodules and although measurable activities of xanthine oxidoreductase and urate oxidase were detected in these nodules, the presence of air greatly increased the levels of both enzymes (Fig. 5). In some cases the pattern of enzyme activity with time was altered by addition of NO;-, but in general, this supplementation of N supply did not substantially increase the level of enzymes in Ar: O2-grown nodules (Fig. 5).
C.A. A t k i n s et al. : N n u t r i t i o n a n d N fixation in
Vigna n o d u l e s
Discussion
Effect of N 2 deficiency on pathways of ammonia assimilation. In a previous study (Atkins et al. J 984) it was demonstrated that early development (up to 18 d after sowing) of cowpea nodules was very similar in air and in Ar: 0 2. Rates of growth and structural development were comparable and, provided that the Ar:O2-treated plants were supplemented with N O j , their non-fixing nodules achieved protein contents comparable to those of air-grown nodules. Subsequently, however, the course of development in Ar: 0 2 diverged markedly from that of normal nodule development in air, with the Ar:O2-treated nodules exhibiting premature senescence at 20 d, and even when plant N deficiency had been relieved by added NO 3, senescence under At: 02 was delayed by only a few days. When developing in air, the functioning symbiosis matured progressively with increasing levels of export of reduced N to the host up to 24 d after sowing (Fig. 4). While reducing markedly the export of N in xylem, culture of nodules in Ar:O 2 did not reduce significantly nitrogenase activity or Ieghaemoglobin development during the first ~8 d after sowing. Thus, absence of N 2 failed to alter the derepression of those plant and bacteroid-associated biochemical systems involed in the establishment of nitrogenase functioning in the developing nodule. In this respect the "effective" and "inoperative" (Ar:Oz-grown) nodules were highly comparable. To a greater or lesser extent all enzymes likely to be associated with the assimilation of ammonia and the synthesis of ureides (Fig. 5) were markedly stimulated by N2, and although the effector may be different in each case, the response was indicative of a regulatory role for either ammonia or products of its assimilation. Not all enzymes of N metabolism showed clear stimulation; aspartate aminotransferase and allantoinase were less sensitive while glutamate oxidoreductase was unaffected (Fig. 5). Furthermore, phosphoenolpyruvate carboxylase, an enzyme which may have a more general anaplerotic role in the carbon metabolism of nodules (Christeller et al. 1977; Cookson et al. 1980), showed a marked stimulation by N 2 (Fig. 5), perhaps indicating that other metabolic functions of the symbiosis were also regulated by the ammonia supply. Except for the enzymes of de-novo purine synthesis, all enzymes of N metabolism assayed were readily detected, albeit at reduced levels, in Ar:O 2grown nodules (Fig. 5). This was not surprising for enzymes such as glutamine synthetase, gluta-
331
mate synthase and aspartate aminotransferase, which catalyse reactions common to most plant tissues (see review by Miflin and Lea 1980), but for the enzymes of purine oxidation, xanthine oxidoreductase and urate oxidase, activities in Ar: 0 2grown nodules were considerably higher than those normally detected in other tissues of cowpea (Herridge et al. 1978) or in tissues and isolated cells of non-leguminous plants (Barankiewicz and Paszkowski 1980; Nguyen and Nato 1983). Although substantial transfer of ammonia from the bacteroid may have resulted in stimulation of the pathways of N metabolism, the high level of transfer associated with an "effective" symbiosis was apparently not necessary for derepression of these enzymes. The response of de-novo purine synthesis to N 2 deficiency was exceptional in that virtually no activity was detected in nodules grown in Ar:O 2 whether or not NO~- was added (Fig. 5). The pathway, measured in tissue extracts by synthesis of labeled inosine monophosphate from [14C]glycine, involves activity of at least eleven separate enzymes (see review by Atkins 1982) and it is conceivable that in Ar:O 2 repression or deletion of just one of these could have prevented activity. Labeled intermediates of the de-novo purine sequence, such as aminoimidazole carboxamide ribonucleotide or formyl-glycinamide ribonucleotide, which might have been expected to accumulate if only a part of the sequence was deleted (Atkins et al. 1982), were however not detected. In view of the fact that the whole metabolic pathway of inosine monophosphate synthesis from phosphoribosylpyrophosphate has been shown to be localized within the plastids of nodule cells (Shelp et al. 1983), it seems likely that the total sequence remained repressed in the absence of a pronounced flux of ammonia from the bacteroids. Interestingly, in other tissues of cowpea, assimilation of ammonia resulting from NO~ reduction does not lead to substantial ureide synthesis but rather to formation of amides and amino acids (Atkins et al. 1980a) as is the case in nodules of "non-ureideproducing" legumes (Atkins 1982). Induction of the ureide pathway in nodules, especially of the plastid-located de-novo purine sequence, may be regarded as a specific feature, like the formation of leghaemoglobin and other special plant proteins (nodulins; Legocki and Verma 1979), of an effective symbiosis. Effect of N 2 deficiency on bacteroid enzymes. The consequent N deficiency associated with AriO 2 treatment resulted in a marked reduction of bacte-
332
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
roid-protein levels even at 14 d after sowing (Atkins et al. 1984), and although addition of NO~ overcame N deficiency and greatly stimulated protein levels in nodules, most of the " e x t r a " protein was formed in the plant cells, with little transfer of N to the bacteroids (Atkins et al. 1984). Despite this N deficiency, nodules from Ar:O2-grown plants showed nitrogenase levels comparable with those in air up to 16 d after sowing and, even after the onset of senescence, marked activity was retained up to 24 d after sowing. In fact, the specific activity of C2H 2 reduction, expressed on a bacteroid-protein basis (calculated from data of Fig. 2 and Atkins etal. 1984), was similar in Ar:Og-grown (5.1 _+0.7 gmol h - 1 rag- 1) and air-grown nodules (4.4_+ 1.9) throughout the experiment. Hydroxybutyrate oxidoreductase activity was markedly stimulated in A r : O z (Fig. 5). Interestingly, increased levels of this bacteroid enzyme have been noted previously in normally senescing nodules of soybean (Stripf and Werner 1978) or following treatments which induced premature nodule senescence (Wong and Evans 1971). Earlier studies using Rhizobium strain CB756 in a number of symbioses with cowpea (Rainbird et al. 1983; Schubert et al. 1978), have inferred the presence of a unidirectional uptake hydrogenase. The very sharp decline in H 2 evolution in both nodule types by 15 or 16 d (Fig. 1), despite continued high rates of C2H 2 reduction, probably reflects the sudden activity of such an enzyme in H 2 uptake. Continued H 2 release in Ar:O2 while the rates declined to zero in air (Fig. 1) indicates, however, that there was considerably less effective recycling of H 2 in A r : O 2 compared with air. Apparently the uptake hydrogenase, though initially induced, was not maintained in the absence of N 2, Thus, although in Ar: 0 2 there was continued utilization of translocated sugar in respiratory metabolism, the efficiency of carbon use by nodules was apparently reduced. In this context it will be interesting to see if the special pathways of electron transport and oxidative phosphorylation, formed in bacteroids under conditions of low 0 2 tension and specifically associated with efficient nitrogenase functioning (Appleby 1969a), are also derepressed at very low levels of N 2.
References
The authors wish to thank P. Sanford for skilled technical assistance. The work was supported by grants from the Australian Research Grants Scheme (to C.A.A.), the Wheat Industry Research Council of Australia (to C.A.A. and J.S.P.) and by a National Science and Engineering Research Council of Canada post-doctoral fellowship (to B.J.S.).
Appleby, C.A. (1969 a) Electron transport systems of Rhizobium japonicum: haemoprotein P-450, other CO-reactive pigments, cytochromes and oxidases in bacteroids from N z fixing root nodules. Biochem. Biophys. Acta 172, 71-87 Appleby, C.A. (1969b) Properties of leghaemoglobin in vivo and its isolation as ferrous oxyleghaemoglobin. Biochim. Biophys. Acta 188, 222-229 Atkins, C.A. (1974) Occurrence and some properties of carbonic anhydrases from legume root nodules. Phytochemistry 13, 93-98 Atkins, C.A. (1982) Ureide metabolism and the significance of ureides in legumes. In: Advances in agricultural microbiology, pp. 211-242, Subba Rao, N.S., ed. Oxford and IBH Publishing Co., New Delhi, India Atkins, C.A., Pate, J.S., Griffiths, G.J., White, S.T. (1980a) Economy of carbon and nitrogen in nodulated and nonnodulated (NO3-grown) cowpea (Vigna unguiculata (L.) Walp.). Plant Physiol. 66, 978-983 Atkins, C.A., Rainbird, R.M., Pate, J.S. (1980b) Evidence for a purine pathway of ureide synthesis in N2-fixing nodules of cowpea (Vigna unguiculata (L.) Walp). Z. Pflanzenphysiol. 97, 249-260 Atkins, C.A., Ritchie, A., Rowe, P.B., McCairns, E., Sauer, D. (1982) De novo purine synthesis in nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp) and soybean (Glycine max L. Merr). Plant Physiol. 70, 55-60 Atkins, C.A., Shelp, B.J., Kuo, J., Peoples, M.B., Pate, J.S. (1984) Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings. Planta 162, 316-326 Barankiewicz, J., Paszkowski, J. (1980) Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves. Biochem. J. 186, 343-350 Christeller, J.T., Laing, W.A., Sutton, W.D. (1977) Carbon dioxide fixation by lupin root nodules. I. Characterisation, association with phosphoenolpyruvate carboxylase, and correlation with nitrogen fixation during nodule development. Plant Physiol. 60, 47-50 Cookson, C., Hughes, H., Coombs, J. (1980) Effects of combined nitrogen on anapleurotic carbon assimilation and bleeding sap composition in PhaseoIus vulgar& L. Planta 148, 429-433 Herridge, D.F., Atkins, C.A., Pate, J.S., Rainbird, R.M. (1978) Allantoin and allantoic acid in the nitrogen economy of the cowpea (Vigna unguiculata (L.) Walp). Plant Physiol. 62, 495-498 Legocki, R.P., Verma, D.P.S. (1979) A nodule-specific plant protein (nodulin-35) from soybean. Science 205, 190-193 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 Miflin, B.J., Lea, P.J. (1980) Ammonia assimilation. In: The biochemistry of plants, vol. 5 : Amino acids and derivatives, pp. 169-202, Miflin, B.J., ed. Academic Press, New York London Newcomb, W. (1981) Nodule morphogenesis and differentiation. Int. Rev. Cytol., Suppl. 13, 247-298 Nguyen, J., Nato, A. (1983) In vitro study of the xanthine dehydrogenase from illuminated or darkened leaves. Physiol. Plant. 59, 73-78 Rainbird, R.M., Atkins, C.A., Pate, J.S., Sanford, P. (1983) Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea. Plant Physiol. 71, 122-127 Robertson, J.G., Farnden, K.J.F., Warburton, M.P., Banks, J.A.M. (1975a) Induction of glutamine synthetase during
C.A. Atkins et al. : N nutrition and N fixation in Vigna nodules
333
nodule development in lupin. Aust. J. Plant Physiol. 2, 265-272 Robertson, J.G., Warburton, M.P., Farnden, K.J.F. (1975b) Induction of glutamate synthase during nodule development in lupin. FEBS Lett. 55, 33-37 Schubert, K.R., Jennings, N.T., Evans, H.J. (1978) Hydrogen reactions of nodulated leguminous plants. II. Effects on dry matter accumulation and nitrogen fixation. Plant Physiol. 61,398-401 Shelp, B.J., Atkins, C.A., Storer, P.J., Canvin, D.T. (1983) Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata (L.) Walp.). Arch. Biochem. Biophys. 224, 429-441
Stripf, R., Werner, D. (1978) Differentiation of Rhizobiumjaponicum. II. Enzymatic activities in bacteroids and plant cytoplasm during the development of nodules of Glycine max. Z. Naturforsch. Teil C 33, 373-381 Sutton, W.D. (1983) Nodule development and senescence. In: Nitrogen fixation, vol. 3: Legumes, pp. 144-212, Broughton, W.J., ed. Clarendon Press, Oxford, UK Wong, P.P., Evans, H.J. (1971) Polyhydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiol. 47, 750-755 Received 2 March; accepted 7 May 1984
