Planta 9 by Springer-Verlag 1977
Planta 134, 17-22 (1977)
Nitrate Effects on the Nodulation of Legumes Inoculated with Nitrate-reductase-deficient Mutants of Rhizobium A.H. Gibson and Janet D. Pagan 9 Divisionof Plant Industry,CSIRO, P.O.B. 1600, Canberra City, A.C.T. 2601, Australia
Abstract. The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (lamol C2H4 g-1 nodule ft. wt. h-1), were both affected by the added nitrate.
Key words: Leguminosae - Nitrate-reductase mutants - Nitrogen fixation - Nodulation - Rhizobium.
Introduction
Nitrate retards the development of nodules on legumes nodulated with Rhizobium strains (Thornton, 1936; Gibson and Nutman, 1960), an effect ascribed to the inhibition of root hair infection (Thornton, 1936; Munns, 1968a). Indolyl-3-acetic acid (IAA), produced by rhizobia from tryptophane in the root exudates, is probably involved in root-hair infection
and nodule initiation (Kefford et al., 1960; Libbenga and Torrey, 1973). This, and the observation that most rhizobia can reduce nitrate to ni;trite, prompted Tanner and Anderson (1963, 1964) to propose that the inhibitory action of nitrate was based upon the catalytic destruction of IAA by nitrite. Support for this hypothesis was provided by studies in which supplemental IAA in the culture medium partially alleviated the inhibitory effects of nitrate on nodulation (Valera and Alexander, 1965; Munns, 1968b). Nitrate is also responsible for decreasing the level of nitrogen fixation by nodulated legumes. This effect of nitrate has been attributed to a diminished Supply of photosynthate available to the nodules, following its utilization in the assimilation of nitrate in the shoots and roots (Wilson and Fred, 1939; Allison, 1939; Small and Leonard, 1969; Oghoghorie and Pate, 1971). However, much of the data pertaining to the decreased supply of photosynthate to nodules could be explained if the added nitrate acted within the nodules to inhibit nitrogen fixation, thereby reducing the role of the nodule as a sink for photosynthate (Gibson, 1976). For example, the inhibition could be caused by nitrite, produced as a consequence of nitrate-reductase activity in the nodules (Cheniae and Evans, 1957; Bergersen, 1961a). Nitrite is an inhibitor of nitrogenase in bacteroids (Rigaud et al., 1973; Kennedy et al., 1975) and in N2-fixing cultures of rhizobia (Pagan et al., 1977). We have isolated nitrate-reductase-deficient mutants of the strain of cowpea rhizobia, 32H1 (Pagan et al., 1977), and of the Rhizobium trifolii strain, TA1. These mutants provide an opportunity to test the Tanner-Anderson hypothesis in that, in the absence of nitrite production, nodule initiation by the mutants should not be retarded by nitrate. Similarly, if the inhibitory effect of added nitrate on nitrogen fixation is caused by nitrite production in the nodules, the nitrogenase activity in nodules produced by the mu-
18
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
tants should be resistant to added nitrate. The following experiments describe the use of these nitrate/reductase/deficient mutants to examine the effects of nitrate on nodule initiation, and on nitrogen fixation, in Macroptilium atropurpureum ( D C . ) U r b . a n d Trifo-
lium subterraneum L.
Materials and Methods Strains of Rhizobium The copwea strain of rhizobia, 32H1, is capable of N 2 fixation in culture (Pagan et al., 1975), and N 2 fixation by nitrate-reductasedeficient mutants of this strain (NR34, NR165, NR200, NR241) is not affected by nitrate (Pagan et al., 1977). In culture, the mutants NR34, NR200 and NR241 were found to have levels of nitrogenase activity comparable with that of the parent strain 32H 1, and were able to establish an effective symbiosis with Macroptilium atropurpureum. However, strain NR 165 had only 20 per cent of the nitrogenase activity of strain 32H1 when grown in culture; furthermore, the NR165-nodules were slower to commence nitrogen fixation than the 32HI-nodules although levels of activity 23 days after inoculation were comparable (Pagan et al., 1977). Although strain 32H1 grew in shaken liquid medium (Bergersen's (1961b) medium with mannitol, NazHPO 4 and Na-glutamate replaced by arabinose, K2HPO4 and NaNO3) containing nitrate as the nitrogen source, the mutant strains did not, indicating that they had also lost their assimilatory nitrate reductase. The Rhizobium trifolii strain TA1, only has nitrate-reductase activity when grown "anaerobically", with nitrate as the terminal electron acceptor. Nitrate-reductase mutants were derived by selection in a manner basically similar to that described for the 32H1 nitrate-reductase-deficient strains (Pagan et al., 1977), using a synthetic medium (Bergersen, 1961 b) in which glutamate was replaced with nitrate (5 raM) (BN) and containing 16.3 mM KC103. The cultures were grown under N2 for 7 days. Only two of the 13 presumptive mutants did not show any nitrate-reductase activity (NT2, NT6), although others had much less activity than similarlytreated cultures of the parent strain, TA1.
Plant Culture Macroptilium atropurpureum cv. Siratro (" Siratro ") (kindly supplied by Dr R.A. Date, CSIRO, Brisbane, Qd., Australia) and Trifolium subterraneum cv. Mount Barker (subterranean clover) (Brunnings Pty Ltd., Melbourne, Vic., Australia) seeds were surface sterilized, germinated on water agar (0.9%) in Petri dishes, and sown into a aluminium-foil-capped/tubes so that only the roots, growing down an agar slope, were inside the tubes (Gibson, 1965; Vincent, 1970, p. 82). After 3 days, seedling nutrient solution (S.N.S. ; Gibson, 1963) was added to within 3 cm of the top of each tube, and the tubes inoculated with a suspension of rhizobia (106 cells/ tube). The plants were grown in a controlled-environment cabinet (Gibson, 1965), with a 14-h daily light period. The light source was a bank of Philips "Reflectatype" fluorescent tubes (PLFM140W/33RS) with 4 linear incandescent tubes (60cm, 20 W), providing 540 pE m - 2 s- 1, as measured by a Lambda Instruments (4421 Superior Street, Lincoln, Nebraska, 68504, U.S.A. quantum sensor. The temperature conditions for Siratro were 27 ~C for the roots, and 30~ (light period)/25~ (dark period) for the shoots. For subterranean clover, the root temperature was 22 ~ and the shoot temperature regime 22/15 ~ C. For the examination of nitrate effects on initial nodulation, sterile solutions of KNO3 were added at the time of inoculation
to provide 0.3, 1.0 or 3.0 mg N/tube (equivalent to 0.7, 2.3 and 7 mM initially). Four days after inoculation, the roots of each plant were examined against a well-illuminated, back-lit, frosted glass screen, and these observations continued until all plants in each treatment were nodulated. Nodule position was recorded and the observation verified the following day. The time taken for plants to nodulate is a very sensitive measure of the effects of different treatments. Nodule counts have meaning only in the short term (2-3 days after the first nodules appear) in this type of investigation as other factors, such as preexisting nodules, the extent of lateral root development, and the ultimate stimulatory effect of nitrate on nodulation, affect the rate at which nodules appear (Gibson and Nutman, 1960; Gibson, 1974). For the study of nitrate effects on nitrogen fixation, inoculated Siratro plants were grown in the absence of nitrate for 23 days before applying the treatments. The population of plants in each strain treatment was ranked according to size and distributed to groups such that the composition of all groups was similar. To each tube, 0, 1 or 3 mg nitrate-N was added and the plants returned to the cabinet. Twenty-four and 48 h later, 5 pairs of roots from each strain x N-level treatment were assayed for nitrogenase activity by the acetylene reduction technique (assay gas mixture 20% 02, 7% C2H2, 73% Ar, in 30ml bottles; incubation time, 30 45 min) at 27 ~ C. Two days after adding nitrate, nodules were removed from all roots after the C2H z reduction assay, and their fresh weight was determined. In one experiment (III), involving 32Hl and NR200, a further assay was made 7 days after adding nitrate. The same methods were used with subterranean clover except that the plants were 33 days old when nitrate was added (2 or 4 mg N/tube) and the assay temperature was 22 ~ C.
Nitrate-reductase Assays Assays on 32H1 and the mutants derived from this strain were made as described previously (Pagan et al., 1977). For strains TA1, NT2 and NT6, the bacteria were grown in yeast-arabinose medium (arabinose replacing mannitol in YMA) until late log phase, washed in phosphate buffer (0.05 mM, pH 7.0) and then suspended in sufficient BN liquid medium to fill 100-ml screw top bottles completely. The tops were tightened and the bottles kept at 30~ C for 3-4 days to permit the development of nitrate reductase, which in TA1 is an inducible enzyme. The cultures were assayed for nitrate reductase in whole cells (Pagan et al., 1977). Ca. 100 mg of fresh nodules were ground in 25 mM Tris-HC1 buffer, pH 7.4, under a stream of N2, and assayed for nitrate reductase activity by the method described by Rigaud et al. (1973). As the assay determines nitrite concentration, it measures both the production of nitrite by the action of nitrate reductase and the presence of any nitrite transported into the nodules from the root system.
Results Nitrate Inhibition o f Initial Nodulation The presence of nitrate delayed the appearance of n o d u l e s o n S i r a t r o p l a n t s i n o c u l a t e d w i t h s t r a i n 32H1 a n d w i t h the n i t r a t e - r e d u c t a s e - d e f i c i e n t m u t a n t s (Table 1). N o d u l e c o u n t s 8 d a y s a f t e r i n o c u l a t i o n v e r i f i e d that the inhibition of nodule initiation applied equally t o t h e p a r e n t s t r a i n a n d t h e m u t a n t s t r a i n s ( T a b l e 1).
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase M u t a n t s of Rhizobium Table 1. The effect of N O 3 - N on the m e a n time to initial nodulation (days from inoculation) and the number of nodules formed 8 days after inoculation (mean per plant) when Siratro plants were inoculated with the cowpea strain of rhizobia, 32H1, and 4 m u t a n t strains derived from strain 32H1
Parameter
Nitrate-N added (mg)
32H1
NR34
Initial nodulation (days)
0 0.3 h0 3.0
6,7 7.0 %4 7.9
6.4 6.8 7.9 8.5
Nodule number (day 8)
0 0.3 1.0 3.0
9,7 8,1 7.5 4,2
8.3 9.5 5.5 1.3
1.s.d. (p=0.05): number, 4.6
NR165
NR200
NR241
7.1 7.3 7.7 8.8
7.3 7.4 7.6 8.8
6.7 7.0 7.8 8.3
10.6 8.1 10.9 0.9
6.2 8.1 4.7 0.3
8.7 9.3 3.1 2.0
time to initial nodulation,
0.9days;
nodule
Table 3. The effect of nitrate on nodule fresh weight (mg/plant) 2 days after adding nitrate in the culture medium In both experiments, the addition of nitrate was made 23 days after inoculation Expt.
Nitrate-N added (mg)
32H1
NR34
NR165
NR241
NR200
I
0 1 3
67.0 59.7 64.7
72.3 61.7 60.7
62.2 62.3 63.7
61.5 55.9 55.9
-
II
0 1 2
60.2 52.7 39.7
58.1 51.2 51.4
70.5 60.8 58.6
56.0 51.4 53.7
66.3 48.5 -
Table 4. Nitrate effects on nodule fresh weight (mg/plant) and nitrogenase specific activity in the nodules (~tmol C z H , g - 1 nodule fr. wt. h - 1) 2 days after nitrate addition
Data pooled over strains in each experiment
Table 2. Nitrogenase activity (Ixmol C2H 4 p l a n t - 1 h - 1) of Siratro plants inoculated with strain 32H1 and 4 nitrate-reductase-negative mutants and supplied with 1 or 3 mg N O ~ - N / t u b e on day 0 Five replicates assayed each day, comprising 2 nodulated roots/ sample (Expt. I) Day
19
Nitrate-N added (mg)
32H1
NR34
NR241
1
0 1 3
1.54 1.00 0.64
1.40 0.82 0.83
1.30 0.90 0.88
1.23 0.86 0.75
2
0 1 3
1.73 1.42 0.99
1.60 1.49 1.21
1.65 1.03 0.91
1.25 0.91 0.70
Expt.
Nitrate-N added (mg)
Nodule weight
Specific activity
mg/plant
% of control
lamol C2H4g-lh
I
0 1 3
66.4 58.4 59.7
100 88 90
22.9 19.5 15.1
100 85 66
II
0 1 2
62.2 52.9 50.9
100 85 82
28.8 27.2 22.2
100 94 77
NR165
The nodulation of subterranean clover inoculated with the two R. trifolii nitrate-reductase-deficient mutants (NT2, NT6) and with the parent strain (TA1) was retarded equally by nitrate.
Inhibition of Nitrogen Fixation by Nitrate Siratro plants nodulated by strains 32H1, NR34, NR165 or NR241 showed a decline in nitrogenase activity 24 h after the addition of nitrate to the culture medium (Table 2). The extent of the decline was similar for the plants in all inoculation treatments, with analyses of variance on the data showing N-level effects (3.0 < 1.0 < 0) but no strain • N-level interaction (/)=0.05). In a further experiment (III, Table 5), plants nodulated by strain NR200 showed a similar response to the addition of nitrate as did those nodulated by 32H1.
% of 1 control
The decline in overall nitrogenase activity on a per-plant basis (Table 2) could be based upon an effect on nodule weight, an effect on the nitrogenase specific activity (lamol CzH4 g-1 nodule fr. wt. h-1) of the nodules, or both. Examination of the nodule weight data derived 2 days after the addition of nitrate (Table 3) showed a consistent inhibitory effect of nitrate on nodule weight in this (I), and a subsequent (II) experiment. Analysis of variance showed that the nitrate effect was significant (p=0.05) and that there was no strain effect. Although the nodule weights are only 10-18% lower in the nitrate treatments than in the controls, nodule-weight determinations on day zero (see Table 5) showed that there was little or no nodule growth in the 2 days after imposing the nitrate treatment. The specific activity of nitrogenase in the nodules was also inhibited by the addition of nitrate in the two experiments, especially at the higher nitrate concentrations (Table 4). To examine recovery from the effects of adding nitrate, plants inoculated with strains 32H1 and NR200 were assayed 7 days after imposition of the treatment. There Were higher levels of nitrogenase activity per plant than found with the earlier assays
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
20
Table 5. The effect of nitrate on the total nitrogenase activity (gmol C2H4 plant- 1 h ~), nodule fresh weight (mg plant- 1), and nitrogenase specific activity of the nodules (lamol C2H4 g-~ nodule fr. wt. h-1.) of plants inoculated with strains 32H1 and NR200 (Experiment III) Nitrate added 23 days after inoculation Parameter
Day Strain 32H1 Control 1.23 1.60 1.66 1.90
Strain NR200
1 mgN
3mgN
-
-
Control
3mgN
-
-
Total activity
0 1 2 7
Nodule weight
0 2 7
53.0 66.5 86.8
52.7 81.7
54.5 67.6
56.6 68.6 105.2
58.0 87.1
58.8 78.4
Specific activity
0 2 7
23.0 25.1 21.4
19.2 26.2
14.3 21.5
30.9 17.6 18.2
16.6 25.8
12.7 18.7
1.29 0.97 2.13
1.39 0.79 1.43
1.71 1.60 1.19 1.91
1 mgN
1.36 0.97 2.26
1.18 0.75 1.47
Table 6. Nitrate effects on nitrogenase activity (pmol C2H4 plant- 1 h - t ) in nodules on subterranean clover inoculated with strain TA1 or 2 mutant strains derived from strain TA1, 1 and 2 days after the addition of nitrate Nitrate-N added (mg)
Day
TA1
NT2
NT6
0
1 2
1.32 2.07
1.26 1.63
1.21 1.56
2.0
1 2
1.06 1.34
0.86 1.34
1.05 1.03
4.0
1 2
1.03 0.95
0.83 0.70
0.94 0.89
(Table 5). The values for the 1 mg N/tube treatment exceeded that for the " n o N " controls (p=0.05; no strain effects). Nodule growth had recommenced after the retardation following nitrate addition, but was still significantly less than that on the controls. Assays for nitrate reductase in the nodules 2 days after adding nitrate failed to detect any nitrite in the nodules formed by the nitrate reductase deficient strains. On the other hand, high levels of nitrate reductase activity ( > 100 gmol NO[ g- t nodule fr. wt. h-1) were found in the nodules formed by strain 32H1, both in the presence and the absence of added nitrate. Subterranean clover plants inoculated with strain TA1, and the two nitrate-reductase-deficient strains derived from strain TA1, showed lower levels of nitrogenase activity 1 and 2 days after the addition of nitrate to the plant culture medium (Table 6). The extent of the depression in activity was similar for
the three strains. Nitrate-reductase assays on suspensions of crushed nodules from plants inoculated with each of the strains, either with or without 4 mg N/ tube, failed to detect any nitrate-reductase activity in 30-min assays. In strain TA1, nitrate-reductase is inducible, developing under conditions of low 02 tension when exposed to nitrate. Examination of nodule weights 2 days after adding nitrate, and of the calculated nitrogenase specific activity of the nodules, failed to give any clear indication as to whether the principal effect was on nodule development or on specific activity; both were affected.
Discussion
The failure to circumvent nitrate inhibition of nodulation by the use of nitrate-reductase-deficient mutants indicates that some modification of the proposal by Tanner and Anderson (1963, 1964) is appropriate. As none of these strains were capable of producing nitrite, the delayed initial nodulation cannot be attributed to catalytic destruction of the IAA by bacteriallyproduced nitrite. Nitrite produced by nitrate-reductase activity in the root tissue may be responsible for destruction of IAA if IAA destruction is the basic cause of the inhibition of nodulation by nitrate. Tanner and Anderson (1964) recognized that internal factors such as the C:N ratio (Wilson and Fred, 1939) or the level of available carbohydrate (Allison, 1939) can also control nodule development, and it is possible that such factors have exerted an overriding effect on the nodulation of the plants examined in this investigation. Whatever the nature of the retardation of nodulation by nitrate, the results (Table 1) indicate that the use of strains deficient in nitrate-reductase activity does not appear to be a feasible way of alleviating this inhibition. Two possible mechanisms have been proposed to explain nitrate-induced depression of nitrogenase activity by rhizobia. Virtanen (1950) st/ggested that nitrite forms an NO-compound with leghaemoglobin, thus destroying its function as an Oz-supplier to the bacteroids. Nitrite is also a potent inhibitor of nitrogenase activity, in bacteroids extracted from nodules (Rigaud et al., 1973), in N2-fixing cultures of rhizobia (Pagan et al., 1977) and of the nitrogenase enzyme in vitro (Kennedy et al., 1975). The presence of nitrate reductase in nodules, and the absence of nitrite reductase (Daniel and Appleby, 1972), could be a source of nitrite that could interfere with nitrogenase activity by affecting leghaemoglobin or the enzyme nitrogenase, separately or simultaneously. The results obtained with the nitrate-reductase mutant strains in this investigation indicate that nei-
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
ther of these forms of inhibition are responsible for the lowered levels of nitrogenase activity when the plants are supplied with nitrate. Neither dissimilatory nor assimilatory nitrate-reductase activity could be detected in the nodules formed by the mutants. Nor could nitrite be detected in these nodules, indicating that there was no translocation of nitrite to the nodules from the roots. Furthermore, N 2 fixation in culture by the nitrate-reductase-defieient mutants is not affected by levels of nitrate which, when supplied in the plant culture medium, depress nitrogenase activity by these strains in nodules (Pagan et al., 1977). All of these observations indicate that the lowered nitrogenase activity in the nodules on plants supplied with nitrate is due to factors other than nitrite inhibition. A less direct effect of nitrate in inhibiting nitrogen fixation could be caused by the translocation of ammonia to the nodules following nitrate-reductase activity in the roots. As nitrogen fixation by nodulated legumes is inhibited less by ammonia than by nitrate at equimolar concentrations (Gibson, 1974), it is unlikely that the nitrate effect is only an effect of ammonia. Furthermore, ammonia does not inhibit nitrogenase activity per se in short-term assays with bacteroids (Bergersen, 1969). Conversely, the results offer some support for the "photosynthate deprivation" hypothesis (Oghoghorie and Pate, 1971). The retardation of nodule development (Table 3) can be interpreted as resulting from a lack of available photosynthate. The higher specific activity of nitrogenase in the nodules on plants supplied with 1 mg N 7 days previously (Table 5) indicates that the inhibition of activity caused by nitrate is temporary, and not based upon permanent damage to the enzyme system or the symbiotic association (Dart and Mercer, 1965) by nitrate or nitrite. If photosynthate deprivation is the principal factor responsible for a decline in nitrogenase activity following the application of nitrate to nodulated plants, the search for strains of Rhizobium able to with stand the effects of combined nitrogen would appear to stand little chance of success. Certainly the use of nitrate-reductase-deficient mutant strains in this investigation has shown that these strains are not able to nodulate the host, nor able to maintain high rates of N 2 fixation, any better than their parent strains when the plants are provided with nitrate.
The authors are grateful to Mrs. M. Ashby, Miss Y. Hort and Miss L. McCurley for their very competent assistance throughout the investigation, and to Drs. J. Denarie, Versailles, and J. Rigaud, Nice, France, for helpful discussions.
21
References Allison, F.E. : Legume nodule development in relation to available energy supply. J. Amer. Soc. Agron. 31, 149 158 (1939) Bergersen, F.J.: Nitrate reductase in soybean root nodules. Biochim. Biophys. Acta 52, 206 207 (1961a) Bergersen, F.J. : The growth of Rhizobium in the synthetic media. Aust. J. biol. Sci. 14, 349 360 (1961b) Bergersen, F.J.: Nitrogen fixation in legume root nodules: biochemical studies with soybean. Proc. Roy. Soc. (Lond.) B 172, 401 416 (1969) Cheniae, G.M., Evans, H.J.: On the relation between nitrogen fixation and nodule nitrate reductase of soybean root nodules. Biochim. Biophys. Acta 26, 654-655 (1957) Daniel, R.M., Appleby, C,A.: Anaerobic-nitrate, symbiotic and aerobic growth of Rhizobium japonieum : effects on cytochrome P450, other baemoproteins, nitrate and nitrite reductases. Biochim. Biophys. Acta 275, 347 354 (1972) Dart, P.J., Mercer, F.V. : The influence of ammonium nitrate on the fine structure of nodules of Medicago tribuloides Desr. and Trifolium subterraneum L. Arch. Mikrobiol. 51, 233-257 (1965) Gibson, A.H. : Physical environment and symbiotic nitrogen fixation. I. The effect of root temperature on recently nodulated Trifolium subterraneum L. plants. Aust. J. biol. Sci. 16, 28-42 (1963) Gibson, A.H. : Physical environment and symbiotic nitrogen fixation. II. Root temperature effects on the relative nitrogen assimilation rate. Aust. J. biol. Sci. 18, 295 310 (1965) Gibson, A.H. : Consideration of the growing legume as a symbiotic association. Proc. Ind. Nat. Sci. Acad. 40B, 741-767 (1974) Gibson, A.H. : Recovery and compensation by nodulated legumes to environmental stress. In: Symbiotic nitrogen fixation in plants, pp. 385-403, Nutman, P.S., ed. Cambridge: Univ. Press 1976 Gibson, A.H., Nutman, P.S. : Studies on the physiology of nodule formation. VII. A reappraisal of the effect of preplanting. Annl Bot. (Lond.) 24, 420-433 (1960) Kefford, N.P., Brockwell, J., Zwar, J.A. : The symbiotic synthesis of auxin by legumes and nodule bacteria and its role in nodule development. Aust. J. biol. 13, 456-467 (1960) Kennedy, I.R., Rigaud, J., Trinchant, J.C. : Nitrate reductase from bacteroids of Rhizobium japonicum: Enzyme characteristics and possible interaction with nitrogen fixation. Biochim. Biophys. Acta 397, 24-35 (1975) Libbenga, K.R., Torrey, J.G.: Hormone-induced endoreduplication prior to mitosis in cultured pea root cortex cells. Amer. J. Bot. 60, 293-299 (1973) Munns, D.N. : Nodulation of Medicago sativa in solution culture. III. Effects of nitrate on root hairs and infection. Plant and Soil 29, 33-47 (1968a) Munns, D.N.: Nodulation of Medicago sativa in solution culture. IV. Effects of indole-3-acetate in relation to acidity and nitrate. P/ant and Soi1 29, 257~62 (1968b) Oghoghorie, C.G.O., Pate, J.S.: The nit/ate stress syndrome of the nodulated field pea (Pisum arvense L.). In: Biological nitrogen fixation in natural and agricultured habitats, pp. 187-202, Lie, T.A., Mulder, E.G., eds. The Hague: Nijhoff 1971 Pagan, J.D., Child, J.J., Scowcroft, W.R., Gibson, A.H. : Nitrogen fixation by Rhizobium cultured on a defined medium. Nature 256, 406-407 (1975) Pagan, J.D., Scowcroft, W.R., Dudman, W.F., Gibson, A.H. : Nitrogen fixation in nitrate-reductase deficient mutants of cultured rhizobia. J. Bacteriol., in press (1977) Rigaud, J., Bergersen, F.J., Turner, G.L., Daniel, R.M.: Nitrate dependent anaerobic acetylene-reduction and nitrogen fixation by soybean bacteroids. J. gen. Microbiol. 77, 137 144 (1973)
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A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
Small, J.G.C., Leonard, O.A. : Translocation of C14-1abelled ph 0tosynthate in nodulated legumes as influenced by nitrate nitrogen. Amer. J. Bot. 56, 187-194 (1969) Tanner, J.W., Anderson, I.C. : An external effect of inorganic nitrogen in root nodulation. Nature 198, 303 304 (1963) Tanner, J.W., Anderson, I.C. : External effect of combined nitrogen on nodulation. Plant Physiol. 39, 1039 1043 (1964) Thornton, H.G.: The action of sodium nitrate upon the infection of lucerne root hairs by nodule bacteria. Proc. Roy. Soc. (Lond.) B 119, 474-492 (1936) Valera, C.L., Alexander, M. : Reversal of nitrate inhibition of nodulation by indolyl-3-acetic acid. Nature 206, 326 (1965)
Vincent, J.M.: A Manual for the practical study of root-nodule bacteria. International Biological Programme Hdbk. No. 15. Oxford: Blackwell Scient. Publ. 1970 Virtanen, A.I. : Microbiology and chemistry of symbiotic nitrogen fixation. Proc. VII Int. Bot. Congr., Stockholm, pp. 156 159 (1950) Wilson, P.W., Fred, E.B.: The carbohydrate-nitrogen relation in legume symbiosis. J. Amer. Soc. Agron. 31, 49%502 (1939)
Received 9 August; accepted 29 September
Planta 134, 17-22 (1977)
Nitrate Effects on the Nodulation of Legumes Inoculated with Nitrate-reductase-deficient Mutants of Rhizobium A.H. Gibson and Janet D. Pagan 9 Divisionof Plant Industry,CSIRO, P.O.B. 1600, Canberra City, A.C.T. 2601, Australia
Abstract. The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (lamol C2H4 g-1 nodule ft. wt. h-1), were both affected by the added nitrate.
Key words: Leguminosae - Nitrate-reductase mutants - Nitrogen fixation - Nodulation - Rhizobium.
Introduction
Nitrate retards the development of nodules on legumes nodulated with Rhizobium strains (Thornton, 1936; Gibson and Nutman, 1960), an effect ascribed to the inhibition of root hair infection (Thornton, 1936; Munns, 1968a). Indolyl-3-acetic acid (IAA), produced by rhizobia from tryptophane in the root exudates, is probably involved in root-hair infection
and nodule initiation (Kefford et al., 1960; Libbenga and Torrey, 1973). This, and the observation that most rhizobia can reduce nitrate to ni;trite, prompted Tanner and Anderson (1963, 1964) to propose that the inhibitory action of nitrate was based upon the catalytic destruction of IAA by nitrite. Support for this hypothesis was provided by studies in which supplemental IAA in the culture medium partially alleviated the inhibitory effects of nitrate on nodulation (Valera and Alexander, 1965; Munns, 1968b). Nitrate is also responsible for decreasing the level of nitrogen fixation by nodulated legumes. This effect of nitrate has been attributed to a diminished Supply of photosynthate available to the nodules, following its utilization in the assimilation of nitrate in the shoots and roots (Wilson and Fred, 1939; Allison, 1939; Small and Leonard, 1969; Oghoghorie and Pate, 1971). However, much of the data pertaining to the decreased supply of photosynthate to nodules could be explained if the added nitrate acted within the nodules to inhibit nitrogen fixation, thereby reducing the role of the nodule as a sink for photosynthate (Gibson, 1976). For example, the inhibition could be caused by nitrite, produced as a consequence of nitrate-reductase activity in the nodules (Cheniae and Evans, 1957; Bergersen, 1961a). Nitrite is an inhibitor of nitrogenase in bacteroids (Rigaud et al., 1973; Kennedy et al., 1975) and in N2-fixing cultures of rhizobia (Pagan et al., 1977). We have isolated nitrate-reductase-deficient mutants of the strain of cowpea rhizobia, 32H1 (Pagan et al., 1977), and of the Rhizobium trifolii strain, TA1. These mutants provide an opportunity to test the Tanner-Anderson hypothesis in that, in the absence of nitrite production, nodule initiation by the mutants should not be retarded by nitrate. Similarly, if the inhibitory effect of added nitrate on nitrogen fixation is caused by nitrite production in the nodules, the nitrogenase activity in nodules produced by the mu-
18
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
tants should be resistant to added nitrate. The following experiments describe the use of these nitrate/reductase/deficient mutants to examine the effects of nitrate on nodule initiation, and on nitrogen fixation, in Macroptilium atropurpureum ( D C . ) U r b . a n d Trifo-
lium subterraneum L.
Materials and Methods Strains of Rhizobium The copwea strain of rhizobia, 32H1, is capable of N 2 fixation in culture (Pagan et al., 1975), and N 2 fixation by nitrate-reductasedeficient mutants of this strain (NR34, NR165, NR200, NR241) is not affected by nitrate (Pagan et al., 1977). In culture, the mutants NR34, NR200 and NR241 were found to have levels of nitrogenase activity comparable with that of the parent strain 32H 1, and were able to establish an effective symbiosis with Macroptilium atropurpureum. However, strain NR 165 had only 20 per cent of the nitrogenase activity of strain 32H1 when grown in culture; furthermore, the NR165-nodules were slower to commence nitrogen fixation than the 32HI-nodules although levels of activity 23 days after inoculation were comparable (Pagan et al., 1977). Although strain 32H1 grew in shaken liquid medium (Bergersen's (1961b) medium with mannitol, NazHPO 4 and Na-glutamate replaced by arabinose, K2HPO4 and NaNO3) containing nitrate as the nitrogen source, the mutant strains did not, indicating that they had also lost their assimilatory nitrate reductase. The Rhizobium trifolii strain TA1, only has nitrate-reductase activity when grown "anaerobically", with nitrate as the terminal electron acceptor. Nitrate-reductase mutants were derived by selection in a manner basically similar to that described for the 32H1 nitrate-reductase-deficient strains (Pagan et al., 1977), using a synthetic medium (Bergersen, 1961 b) in which glutamate was replaced with nitrate (5 raM) (BN) and containing 16.3 mM KC103. The cultures were grown under N2 for 7 days. Only two of the 13 presumptive mutants did not show any nitrate-reductase activity (NT2, NT6), although others had much less activity than similarlytreated cultures of the parent strain, TA1.
Plant Culture Macroptilium atropurpureum cv. Siratro (" Siratro ") (kindly supplied by Dr R.A. Date, CSIRO, Brisbane, Qd., Australia) and Trifolium subterraneum cv. Mount Barker (subterranean clover) (Brunnings Pty Ltd., Melbourne, Vic., Australia) seeds were surface sterilized, germinated on water agar (0.9%) in Petri dishes, and sown into a aluminium-foil-capped/tubes so that only the roots, growing down an agar slope, were inside the tubes (Gibson, 1965; Vincent, 1970, p. 82). After 3 days, seedling nutrient solution (S.N.S. ; Gibson, 1963) was added to within 3 cm of the top of each tube, and the tubes inoculated with a suspension of rhizobia (106 cells/ tube). The plants were grown in a controlled-environment cabinet (Gibson, 1965), with a 14-h daily light period. The light source was a bank of Philips "Reflectatype" fluorescent tubes (PLFM140W/33RS) with 4 linear incandescent tubes (60cm, 20 W), providing 540 pE m - 2 s- 1, as measured by a Lambda Instruments (4421 Superior Street, Lincoln, Nebraska, 68504, U.S.A. quantum sensor. The temperature conditions for Siratro were 27 ~C for the roots, and 30~ (light period)/25~ (dark period) for the shoots. For subterranean clover, the root temperature was 22 ~ and the shoot temperature regime 22/15 ~ C. For the examination of nitrate effects on initial nodulation, sterile solutions of KNO3 were added at the time of inoculation
to provide 0.3, 1.0 or 3.0 mg N/tube (equivalent to 0.7, 2.3 and 7 mM initially). Four days after inoculation, the roots of each plant were examined against a well-illuminated, back-lit, frosted glass screen, and these observations continued until all plants in each treatment were nodulated. Nodule position was recorded and the observation verified the following day. The time taken for plants to nodulate is a very sensitive measure of the effects of different treatments. Nodule counts have meaning only in the short term (2-3 days after the first nodules appear) in this type of investigation as other factors, such as preexisting nodules, the extent of lateral root development, and the ultimate stimulatory effect of nitrate on nodulation, affect the rate at which nodules appear (Gibson and Nutman, 1960; Gibson, 1974). For the study of nitrate effects on nitrogen fixation, inoculated Siratro plants were grown in the absence of nitrate for 23 days before applying the treatments. The population of plants in each strain treatment was ranked according to size and distributed to groups such that the composition of all groups was similar. To each tube, 0, 1 or 3 mg nitrate-N was added and the plants returned to the cabinet. Twenty-four and 48 h later, 5 pairs of roots from each strain x N-level treatment were assayed for nitrogenase activity by the acetylene reduction technique (assay gas mixture 20% 02, 7% C2H2, 73% Ar, in 30ml bottles; incubation time, 30 45 min) at 27 ~ C. Two days after adding nitrate, nodules were removed from all roots after the C2H z reduction assay, and their fresh weight was determined. In one experiment (III), involving 32Hl and NR200, a further assay was made 7 days after adding nitrate. The same methods were used with subterranean clover except that the plants were 33 days old when nitrate was added (2 or 4 mg N/tube) and the assay temperature was 22 ~ C.
Nitrate-reductase Assays Assays on 32H1 and the mutants derived from this strain were made as described previously (Pagan et al., 1977). For strains TA1, NT2 and NT6, the bacteria were grown in yeast-arabinose medium (arabinose replacing mannitol in YMA) until late log phase, washed in phosphate buffer (0.05 mM, pH 7.0) and then suspended in sufficient BN liquid medium to fill 100-ml screw top bottles completely. The tops were tightened and the bottles kept at 30~ C for 3-4 days to permit the development of nitrate reductase, which in TA1 is an inducible enzyme. The cultures were assayed for nitrate reductase in whole cells (Pagan et al., 1977). Ca. 100 mg of fresh nodules were ground in 25 mM Tris-HC1 buffer, pH 7.4, under a stream of N2, and assayed for nitrate reductase activity by the method described by Rigaud et al. (1973). As the assay determines nitrite concentration, it measures both the production of nitrite by the action of nitrate reductase and the presence of any nitrite transported into the nodules from the root system.
Results Nitrate Inhibition o f Initial Nodulation The presence of nitrate delayed the appearance of n o d u l e s o n S i r a t r o p l a n t s i n o c u l a t e d w i t h s t r a i n 32H1 a n d w i t h the n i t r a t e - r e d u c t a s e - d e f i c i e n t m u t a n t s (Table 1). N o d u l e c o u n t s 8 d a y s a f t e r i n o c u l a t i o n v e r i f i e d that the inhibition of nodule initiation applied equally t o t h e p a r e n t s t r a i n a n d t h e m u t a n t s t r a i n s ( T a b l e 1).
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase M u t a n t s of Rhizobium Table 1. The effect of N O 3 - N on the m e a n time to initial nodulation (days from inoculation) and the number of nodules formed 8 days after inoculation (mean per plant) when Siratro plants were inoculated with the cowpea strain of rhizobia, 32H1, and 4 m u t a n t strains derived from strain 32H1
Parameter
Nitrate-N added (mg)
32H1
NR34
Initial nodulation (days)
0 0.3 h0 3.0
6,7 7.0 %4 7.9
6.4 6.8 7.9 8.5
Nodule number (day 8)
0 0.3 1.0 3.0
9,7 8,1 7.5 4,2
8.3 9.5 5.5 1.3
1.s.d. (p=0.05): number, 4.6
NR165
NR200
NR241
7.1 7.3 7.7 8.8
7.3 7.4 7.6 8.8
6.7 7.0 7.8 8.3
10.6 8.1 10.9 0.9
6.2 8.1 4.7 0.3
8.7 9.3 3.1 2.0
time to initial nodulation,
0.9days;
nodule
Table 3. The effect of nitrate on nodule fresh weight (mg/plant) 2 days after adding nitrate in the culture medium In both experiments, the addition of nitrate was made 23 days after inoculation Expt.
Nitrate-N added (mg)
32H1
NR34
NR165
NR241
NR200
I
0 1 3
67.0 59.7 64.7
72.3 61.7 60.7
62.2 62.3 63.7
61.5 55.9 55.9
-
II
0 1 2
60.2 52.7 39.7
58.1 51.2 51.4
70.5 60.8 58.6
56.0 51.4 53.7
66.3 48.5 -
Table 4. Nitrate effects on nodule fresh weight (mg/plant) and nitrogenase specific activity in the nodules (~tmol C z H , g - 1 nodule fr. wt. h - 1) 2 days after nitrate addition
Data pooled over strains in each experiment
Table 2. Nitrogenase activity (Ixmol C2H 4 p l a n t - 1 h - 1) of Siratro plants inoculated with strain 32H1 and 4 nitrate-reductase-negative mutants and supplied with 1 or 3 mg N O ~ - N / t u b e on day 0 Five replicates assayed each day, comprising 2 nodulated roots/ sample (Expt. I) Day
19
Nitrate-N added (mg)
32H1
NR34
NR241
1
0 1 3
1.54 1.00 0.64
1.40 0.82 0.83
1.30 0.90 0.88
1.23 0.86 0.75
2
0 1 3
1.73 1.42 0.99
1.60 1.49 1.21
1.65 1.03 0.91
1.25 0.91 0.70
Expt.
Nitrate-N added (mg)
Nodule weight
Specific activity
mg/plant
% of control
lamol C2H4g-lh
I
0 1 3
66.4 58.4 59.7
100 88 90
22.9 19.5 15.1
100 85 66
II
0 1 2
62.2 52.9 50.9
100 85 82
28.8 27.2 22.2
100 94 77
NR165
The nodulation of subterranean clover inoculated with the two R. trifolii nitrate-reductase-deficient mutants (NT2, NT6) and with the parent strain (TA1) was retarded equally by nitrate.
Inhibition of Nitrogen Fixation by Nitrate Siratro plants nodulated by strains 32H1, NR34, NR165 or NR241 showed a decline in nitrogenase activity 24 h after the addition of nitrate to the culture medium (Table 2). The extent of the decline was similar for the plants in all inoculation treatments, with analyses of variance on the data showing N-level effects (3.0 < 1.0 < 0) but no strain • N-level interaction (/)=0.05). In a further experiment (III, Table 5), plants nodulated by strain NR200 showed a similar response to the addition of nitrate as did those nodulated by 32H1.
% of 1 control
The decline in overall nitrogenase activity on a per-plant basis (Table 2) could be based upon an effect on nodule weight, an effect on the nitrogenase specific activity (lamol CzH4 g-1 nodule fr. wt. h-1) of the nodules, or both. Examination of the nodule weight data derived 2 days after the addition of nitrate (Table 3) showed a consistent inhibitory effect of nitrate on nodule weight in this (I), and a subsequent (II) experiment. Analysis of variance showed that the nitrate effect was significant (p=0.05) and that there was no strain effect. Although the nodule weights are only 10-18% lower in the nitrate treatments than in the controls, nodule-weight determinations on day zero (see Table 5) showed that there was little or no nodule growth in the 2 days after imposing the nitrate treatment. The specific activity of nitrogenase in the nodules was also inhibited by the addition of nitrate in the two experiments, especially at the higher nitrate concentrations (Table 4). To examine recovery from the effects of adding nitrate, plants inoculated with strains 32H1 and NR200 were assayed 7 days after imposition of the treatment. There Were higher levels of nitrogenase activity per plant than found with the earlier assays
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
20
Table 5. The effect of nitrate on the total nitrogenase activity (gmol C2H4 plant- 1 h ~), nodule fresh weight (mg plant- 1), and nitrogenase specific activity of the nodules (lamol C2H4 g-~ nodule fr. wt. h-1.) of plants inoculated with strains 32H1 and NR200 (Experiment III) Nitrate added 23 days after inoculation Parameter
Day Strain 32H1 Control 1.23 1.60 1.66 1.90
Strain NR200
1 mgN
3mgN
-
-
Control
3mgN
-
-
Total activity
0 1 2 7
Nodule weight
0 2 7
53.0 66.5 86.8
52.7 81.7
54.5 67.6
56.6 68.6 105.2
58.0 87.1
58.8 78.4
Specific activity
0 2 7
23.0 25.1 21.4
19.2 26.2
14.3 21.5
30.9 17.6 18.2
16.6 25.8
12.7 18.7
1.29 0.97 2.13
1.39 0.79 1.43
1.71 1.60 1.19 1.91
1 mgN
1.36 0.97 2.26
1.18 0.75 1.47
Table 6. Nitrate effects on nitrogenase activity (pmol C2H4 plant- 1 h - t ) in nodules on subterranean clover inoculated with strain TA1 or 2 mutant strains derived from strain TA1, 1 and 2 days after the addition of nitrate Nitrate-N added (mg)
Day
TA1
NT2
NT6
0
1 2
1.32 2.07
1.26 1.63
1.21 1.56
2.0
1 2
1.06 1.34
0.86 1.34
1.05 1.03
4.0
1 2
1.03 0.95
0.83 0.70
0.94 0.89
(Table 5). The values for the 1 mg N/tube treatment exceeded that for the " n o N " controls (p=0.05; no strain effects). Nodule growth had recommenced after the retardation following nitrate addition, but was still significantly less than that on the controls. Assays for nitrate reductase in the nodules 2 days after adding nitrate failed to detect any nitrite in the nodules formed by the nitrate reductase deficient strains. On the other hand, high levels of nitrate reductase activity ( > 100 gmol NO[ g- t nodule fr. wt. h-1) were found in the nodules formed by strain 32H1, both in the presence and the absence of added nitrate. Subterranean clover plants inoculated with strain TA1, and the two nitrate-reductase-deficient strains derived from strain TA1, showed lower levels of nitrogenase activity 1 and 2 days after the addition of nitrate to the plant culture medium (Table 6). The extent of the depression in activity was similar for
the three strains. Nitrate-reductase assays on suspensions of crushed nodules from plants inoculated with each of the strains, either with or without 4 mg N/ tube, failed to detect any nitrate-reductase activity in 30-min assays. In strain TA1, nitrate-reductase is inducible, developing under conditions of low 02 tension when exposed to nitrate. Examination of nodule weights 2 days after adding nitrate, and of the calculated nitrogenase specific activity of the nodules, failed to give any clear indication as to whether the principal effect was on nodule development or on specific activity; both were affected.
Discussion
The failure to circumvent nitrate inhibition of nodulation by the use of nitrate-reductase-deficient mutants indicates that some modification of the proposal by Tanner and Anderson (1963, 1964) is appropriate. As none of these strains were capable of producing nitrite, the delayed initial nodulation cannot be attributed to catalytic destruction of the IAA by bacteriallyproduced nitrite. Nitrite produced by nitrate-reductase activity in the root tissue may be responsible for destruction of IAA if IAA destruction is the basic cause of the inhibition of nodulation by nitrate. Tanner and Anderson (1964) recognized that internal factors such as the C:N ratio (Wilson and Fred, 1939) or the level of available carbohydrate (Allison, 1939) can also control nodule development, and it is possible that such factors have exerted an overriding effect on the nodulation of the plants examined in this investigation. Whatever the nature of the retardation of nodulation by nitrate, the results (Table 1) indicate that the use of strains deficient in nitrate-reductase activity does not appear to be a feasible way of alleviating this inhibition. Two possible mechanisms have been proposed to explain nitrate-induced depression of nitrogenase activity by rhizobia. Virtanen (1950) st/ggested that nitrite forms an NO-compound with leghaemoglobin, thus destroying its function as an Oz-supplier to the bacteroids. Nitrite is also a potent inhibitor of nitrogenase activity, in bacteroids extracted from nodules (Rigaud et al., 1973), in N2-fixing cultures of rhizobia (Pagan et al., 1977) and of the nitrogenase enzyme in vitro (Kennedy et al., 1975). The presence of nitrate reductase in nodules, and the absence of nitrite reductase (Daniel and Appleby, 1972), could be a source of nitrite that could interfere with nitrogenase activity by affecting leghaemoglobin or the enzyme nitrogenase, separately or simultaneously. The results obtained with the nitrate-reductase mutant strains in this investigation indicate that nei-
A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
ther of these forms of inhibition are responsible for the lowered levels of nitrogenase activity when the plants are supplied with nitrate. Neither dissimilatory nor assimilatory nitrate-reductase activity could be detected in the nodules formed by the mutants. Nor could nitrite be detected in these nodules, indicating that there was no translocation of nitrite to the nodules from the roots. Furthermore, N 2 fixation in culture by the nitrate-reductase-defieient mutants is not affected by levels of nitrate which, when supplied in the plant culture medium, depress nitrogenase activity by these strains in nodules (Pagan et al., 1977). All of these observations indicate that the lowered nitrogenase activity in the nodules on plants supplied with nitrate is due to factors other than nitrite inhibition. A less direct effect of nitrate in inhibiting nitrogen fixation could be caused by the translocation of ammonia to the nodules following nitrate-reductase activity in the roots. As nitrogen fixation by nodulated legumes is inhibited less by ammonia than by nitrate at equimolar concentrations (Gibson, 1974), it is unlikely that the nitrate effect is only an effect of ammonia. Furthermore, ammonia does not inhibit nitrogenase activity per se in short-term assays with bacteroids (Bergersen, 1969). Conversely, the results offer some support for the "photosynthate deprivation" hypothesis (Oghoghorie and Pate, 1971). The retardation of nodule development (Table 3) can be interpreted as resulting from a lack of available photosynthate. The higher specific activity of nitrogenase in the nodules on plants supplied with 1 mg N 7 days previously (Table 5) indicates that the inhibition of activity caused by nitrate is temporary, and not based upon permanent damage to the enzyme system or the symbiotic association (Dart and Mercer, 1965) by nitrate or nitrite. If photosynthate deprivation is the principal factor responsible for a decline in nitrogenase activity following the application of nitrate to nodulated plants, the search for strains of Rhizobium able to with stand the effects of combined nitrogen would appear to stand little chance of success. Certainly the use of nitrate-reductase-deficient mutant strains in this investigation has shown that these strains are not able to nodulate the host, nor able to maintain high rates of N 2 fixation, any better than their parent strains when the plants are provided with nitrate.
The authors are grateful to Mrs. M. Ashby, Miss Y. Hort and Miss L. McCurley for their very competent assistance throughout the investigation, and to Drs. J. Denarie, Versailles, and J. Rigaud, Nice, France, for helpful discussions.
21
References Allison, F.E. : Legume nodule development in relation to available energy supply. J. Amer. Soc. Agron. 31, 149 158 (1939) Bergersen, F.J.: Nitrate reductase in soybean root nodules. Biochim. Biophys. Acta 52, 206 207 (1961a) Bergersen, F.J. : The growth of Rhizobium in the synthetic media. Aust. J. biol. Sci. 14, 349 360 (1961b) Bergersen, F.J.: Nitrogen fixation in legume root nodules: biochemical studies with soybean. Proc. Roy. Soc. (Lond.) B 172, 401 416 (1969) Cheniae, G.M., Evans, H.J.: On the relation between nitrogen fixation and nodule nitrate reductase of soybean root nodules. Biochim. Biophys. Acta 26, 654-655 (1957) Daniel, R.M., Appleby, C,A.: Anaerobic-nitrate, symbiotic and aerobic growth of Rhizobium japonieum : effects on cytochrome P450, other baemoproteins, nitrate and nitrite reductases. Biochim. Biophys. Acta 275, 347 354 (1972) Dart, P.J., Mercer, F.V. : The influence of ammonium nitrate on the fine structure of nodules of Medicago tribuloides Desr. and Trifolium subterraneum L. Arch. Mikrobiol. 51, 233-257 (1965) Gibson, A.H. : Physical environment and symbiotic nitrogen fixation. I. The effect of root temperature on recently nodulated Trifolium subterraneum L. plants. Aust. J. biol. Sci. 16, 28-42 (1963) Gibson, A.H. : Physical environment and symbiotic nitrogen fixation. II. Root temperature effects on the relative nitrogen assimilation rate. Aust. J. biol. Sci. 18, 295 310 (1965) Gibson, A.H. : Consideration of the growing legume as a symbiotic association. Proc. Ind. Nat. Sci. Acad. 40B, 741-767 (1974) Gibson, A.H. : Recovery and compensation by nodulated legumes to environmental stress. In: Symbiotic nitrogen fixation in plants, pp. 385-403, Nutman, P.S., ed. Cambridge: Univ. Press 1976 Gibson, A.H., Nutman, P.S. : Studies on the physiology of nodule formation. VII. A reappraisal of the effect of preplanting. Annl Bot. (Lond.) 24, 420-433 (1960) Kefford, N.P., Brockwell, J., Zwar, J.A. : The symbiotic synthesis of auxin by legumes and nodule bacteria and its role in nodule development. Aust. J. biol. 13, 456-467 (1960) Kennedy, I.R., Rigaud, J., Trinchant, J.C. : Nitrate reductase from bacteroids of Rhizobium japonicum: Enzyme characteristics and possible interaction with nitrogen fixation. Biochim. Biophys. Acta 397, 24-35 (1975) Libbenga, K.R., Torrey, J.G.: Hormone-induced endoreduplication prior to mitosis in cultured pea root cortex cells. Amer. J. Bot. 60, 293-299 (1973) Munns, D.N. : Nodulation of Medicago sativa in solution culture. III. Effects of nitrate on root hairs and infection. Plant and Soil 29, 33-47 (1968a) Munns, D.N.: Nodulation of Medicago sativa in solution culture. IV. Effects of indole-3-acetate in relation to acidity and nitrate. P/ant and Soi1 29, 257~62 (1968b) Oghoghorie, C.G.O., Pate, J.S.: The nit/ate stress syndrome of the nodulated field pea (Pisum arvense L.). In: Biological nitrogen fixation in natural and agricultured habitats, pp. 187-202, Lie, T.A., Mulder, E.G., eds. The Hague: Nijhoff 1971 Pagan, J.D., Child, J.J., Scowcroft, W.R., Gibson, A.H. : Nitrogen fixation by Rhizobium cultured on a defined medium. Nature 256, 406-407 (1975) Pagan, J.D., Scowcroft, W.R., Dudman, W.F., Gibson, A.H. : Nitrogen fixation in nitrate-reductase deficient mutants of cultured rhizobia. J. Bacteriol., in press (1977) Rigaud, J., Bergersen, F.J., Turner, G.L., Daniel, R.M.: Nitrate dependent anaerobic acetylene-reduction and nitrogen fixation by soybean bacteroids. J. gen. Microbiol. 77, 137 144 (1973)
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A.H. Gibson and J.D. Pagan: Nodulation by Nitrate-reductase Mutants of Rhizobium
Small, J.G.C., Leonard, O.A. : Translocation of C14-1abelled ph 0tosynthate in nodulated legumes as influenced by nitrate nitrogen. Amer. J. Bot. 56, 187-194 (1969) Tanner, J.W., Anderson, I.C. : An external effect of inorganic nitrogen in root nodulation. Nature 198, 303 304 (1963) Tanner, J.W., Anderson, I.C. : External effect of combined nitrogen on nodulation. Plant Physiol. 39, 1039 1043 (1964) Thornton, H.G.: The action of sodium nitrate upon the infection of lucerne root hairs by nodule bacteria. Proc. Roy. Soc. (Lond.) B 119, 474-492 (1936) Valera, C.L., Alexander, M. : Reversal of nitrate inhibition of nodulation by indolyl-3-acetic acid. Nature 206, 326 (1965)
Vincent, J.M.: A Manual for the practical study of root-nodule bacteria. International Biological Programme Hdbk. No. 15. Oxford: Blackwell Scient. Publ. 1970 Virtanen, A.I. : Microbiology and chemistry of symbiotic nitrogen fixation. Proc. VII Int. Bot. Congr., Stockholm, pp. 156 159 (1950) Wilson, P.W., Fred, E.B.: The carbohydrate-nitrogen relation in legume symbiosis. J. Amer. Soc. Agron. 31, 49%502 (1939)
Received 9 August; accepted 29 September
