Hanta
Planta (Beri.) 129, 1 - 6 (I976)
9 by Springer-Verlag 1976
Evidence from Inhibitor Studies that the Endophyte Synthesises Nitrogenase in the Root Nodules of Alnus glutinosa L. Gaertn.* R.A. Skeffington** and W.D.P. Stewart Department of Biological Sciences, University of Dundee, Dundee, DD1 4 HN, U.K.
Summary. Nitrogenase activity (acetylene reduction assay) in the nodulated non-leguminous angiosperm Alnus glutinosa is inhibited within minutes when plants are exposed to a gas phase containing 90% oxygen. On returning the plants to air, nitrogenase activity recovers within a few hours, both in the presence of cycloheximide, which inhibits protein synthesis on 80 S (eukaryotic) ribosomes, and in the absence of inhibitor. When chloramphenicol, which inhibits protein synthesis on 70 S (prokaryotic) ribosomes, is added instead ofcycloheximide, recovery from oxygen inhibition does not occur, or occurs only slowly. The effects of chloramphenicol are specific to the D-threo-isomer which indicates a direct inhibition of protein synthesis. Erythromycin has a similar effect to chloramphenicol. Protein biosynthesis in non-nodulated roots is inhibited by cycloheximide but not by chloramphenicol. The results are interpreted as evidence that the nitrogenase within Alnus glutinosa root nodules is synthesised by the microbial symbiont.
Introduction
Over a hundred species of nodulated non-leguminous plants are known to fix N2 (Bond, 1974). They do so only in symbiosis with a micro-organism whose identity has never been established convincingly, but which is thought to be an actinomycete. In the legumeRhizobiurn symbiosis, it is known from nodule fractionation experiments (see e.g. Evans and Phillips, 1975) that the nitrogenase is located in the nodule bacteroids, and there is also recent evidence that it A b b r e v i a t i o n s . CAP = chloramphenicol; CH = cycloheximide. ** Present address : Department of Botany, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. *
is genetically coded for by the microbial symbiont, even if this information is usually repressed in the free-living state (see Phillips et al., 1973; Dunican and Tierney, 1974; Kurz and LaRue, t975; McComb et al., 1975; Pagan et aL, 1975). However, it is not possible to locate the site of nitrogenase activity in nonlegume root nodules using fractionation experiments, since consistently high nitrogen-fixing preparations of such nodules have not yet been obtained, neither has the microbial symbiont been isolated with certainty in the free-living state, let alone shown to fix nitrogen. In an attempt to decide whether the nitrogenase in the root nodules of Alnus glutinosa is synthesised by the prokaryotic or eukaryotic symbiont, we have used a different approach to those already tried. That is, we have inhibited the nitrogenase in intact root nodules irreversibly by adding high levels of oxygen (see Dalton and Mortenson, 1972) and then followed nitrogenase resynthesis in the presence of inhibitors of protein biosynthesis specific to either prokaryotes or eukaryotes. The inhibitors used were chloramphenicol (CAP) which inhibits protein biosynthesis on prokaryotic type (70 S) ribosomes only and cycloheximide (CH) which inhibits this activity on eukaryotic type (80 S) ribosomes only (see Boulter et aL, 1972). In the studies using CAP, both the D-threo and L-threo isomers exert various inhibitory effects on general cell metabolism, but the D-threo isomer is the only one which, in addition, exerts a specific effect on protein biosynthesis (Ellis, 1963). This provides a method of distinguishing whether an observed effect of CAP is due to an inhibition of protein biosynthesis or not. In the case of CH, it has also to be shown that any in vivo effect is specifically on protein biosynthesis and not on some other metabolic process (see e.g. Ellis, 1963; Macdonald and Ellis, 1969). In addition, any effect on protein biosynthesis must be demonstrated directly.
2
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
Materials and Methods Culture of Plants. Seed of Alnus glutinosa L. Gaertn. was collected in the east of Scotland, air dried, and stored in Petri dishes at a temperature of 4 ~ C and a relative humidity of 40%. The seed was germinated when required on moist sterile filter paper in Petri dishes after first being surface sterilized by immersion in 10% sodium hypochlorite for 15 min followed by several washes in sterile distilled water. The dishes were kept in the light at 20 ~ C. About 14% of the seeds germinated_ After ten days, the seedlings were transferred to polythene trays containing one quarter-strength nitrogen-free Crone's solution (Bond, 1951) solidified with 0.8 % (w/v) agar. These were maintained in growth cabinets under a 16 h:8 h light-dark regime, with a day temperature of 22 ~ C, a night temperature of 18~ C and an illuminance of 7,000 lux during the light period. Once the first true leaf appeared, the root system of each plant was treated with 0.5 ml of a suspension of crushed Alnus root nodules, which contained approximately 10 g fresh weight of nodules per 100 ml of distilled water. Nodules appeared on all plants within two weeks, and after a further 2 4 weeks growth in the agar trays, the plants were used for experimentation. Preparation of Plants for Experiments. Uniform batches of plants, aged 6-8 weeks, were selected for each experiment. Each plant was placed in a sterilised test-tube containing 2.5 ml of nitrogen-free full strength Crone's solution. The plants were supported by lightly tying the stem to a polythene strip placed in the tube. The bottom of each tube around the roots was painted black and the top closed With cotton wool, except during the acetylene reduction assays (see below) when a serum stopper replaced the cotton wool. The plants were subjected to the various inhibitor treatments 7 days after transfer to the test-tubes. Treatment of Plants with Inhibitors and Oxygen. D-threo chloramphenicol, erythromycin and cycloheximide were purchased from Sigma Ltd., London: L-threo chloramphenicol was a gift from Dr. R.E. Bowman, Parke-Davis Ltd., Pontypool. The inhibitors were dissolved freshly in Crone's solution before use. At the start of the differential treatments, plants were selected at random and their culture solution replaced by the inhibitor solution. All solutions, including those in the control tubes, were renewed every 3 days. Plants were treated with oxygen by evacuating the sealed test-tubes containing the plants and then adding 90% 02 in air to atmospheric pressure. After exposure of the plants to this gas phase for 100 min, air was re-admitted and assays for acetylene reduction were carried out at intervals thereafter. Acetylene Reduction Assays. These were carried out in each test-tube, under a gas phase of 12% (v/v) acetylene in air. Acetylene reduction was followed in 4 plants for each treatment over the whole experimental period. After a 30 min incubation period in the presence of acetylene, a sample of the gas phase was withdrawn, without terminating the reaction, and assayed for acetylene reduction by gas chromatography (Stewart et al., 1967). At the end of the experiment, the nodules were excised from the plants and a check for free-living nitrogen-fixing contaminants (which were never found) was carried out by assaying the remainder of each plant for a capacity to reduce acetylene to ethylene. The Measurement of Protein Biosynthesis in Roots and Nodules. In these experiments, the plants were treated with inhibitor (0.3 mM CAP and 30 gM CH) followed 24 h later by 90% 02 for 100 rain to inactivate the nitrogenase. Immediately after the oxygen treatment, each intact plant was given 26 gl of 1.66 mM NaH14COs (Radiochemical Centre, Amersham), containing a total of 2.6 pCi 14C, for a period of 42 h. The pH of the culture solution did not change significantly from 5.5 during this treatment. The protein was then extracted separately from the roots and from the nodules (Kennell, 1967) and the total dry weight of extracted matter was also determined. An aliquot of the final solu-
tion was taken for protein determination (Lowry et al., 1951) after sotubilisation of the precipitate with 1 N NaOH. The mean protein content of the precipitates ranged from 80 105%. Radioactivity incorporated into each sample was measured by scintillation counting using the scintillant of Patterson and Greene (1965) and a Tracerlab Corumatic 200 liquid scintillation counter. Quenching was corrected for by the channels ratio method using aniline as standard quenching agent. The counting efficiency was approximately 40%.
Results
1. The Effects of Chloramphenicol and Cycloheximide on Nitrogenase Activity The effects of CAP and CH on nitrogenase activity were checked by adding concentrations of inhibitors reported previously to inhibit prokaryotic (0.5 mM CAP) and eukaryotic (36 pM CH) protein synthesis (Ellis and Macdonald, 1967; Macdonald and Ellis, 1969). The results (Fig. 1) show that CAP abolishes nitrogenase activity almost completely and irreversibly within 48 h. With CH, on the other hand, there is an initial inhibition of acetylene reduction, possibly due to an effect on general cell metabolism, but activity then recovers fairly quickly to its original level. These results show that CAP, the inhibitor of protein synthesis in prokaryotes, is a more effective and persistent inhibitor of nitrogenase activity in intact nodules than is CH, the inhibitor of protein synthesis in eukaryotes.
2. The Effects of Chloramphenicol and Cycloheximide on Recovery of Nitrogenase Activity from Oxygen Inhibition In this experiment, the inhibitors were added, followed by 90% 02 for 100 min to specifically inactivate the
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R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
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nitrogenase, and then recovery of nitrogenase activity was monitored after removal of 02. The results (Fig. 2) show that there is some decline in nitrogenase activity when the inhibitors are added, although the differences between the three treatments (CAP, CH, and untreated control) are not significant at the P--0.05 level. When oxygen is added, nitrogenase activity is almost totally abolished within 5 min in all treatments. On removing the oxygen, nitrogenase activity in the untreated control and in the CH-treated series recovers fairly rapidly and there is no significant difference between these two treatments. However nitrogenase activity recovers more slowly in the CAP-treated plants and throughout the experimental period remains significantly lower than in the other two treatments. These data show that when nitrogenase is inhibited specifically by oxygen and the oxygen then removed, nitrogenase activity recovers rapidly in the presence of the inhibitor of eukaryotic protein synthesis (CH), but does not recover to the same extent in the presence of CAP which inhibits prokaryotic protein synthesis.
3. The Effects of Different Optical Isomers of Chloramphenicol on Nitrogenase Activity These experiments provide evidence that the inhibitory effects of CAP on nitrogenase activity noted in Fig. 1 and Fig. 2 are probably due to an inhibition of protein synthesis. As discussed earlier, protein synthesis in prokaryotes is inhibited only by the D-threo
Fig. 3. The effects of oxygen (90% for 100 rain) on nitrogenase activity by intact plants of Alnus glutinosa treated with 0.3 mM Dthreo CAP ( e - e ) and 0.3 mM L-threo CAP (=-,,), or untreated (A-A). From the point of removal of oxygen, the recovery of D-threo CAP-treated plants differs significantly from the other two treatments (P=0.05 level). The latter treatments do not differ significantly form each other except at the last sampling time. Arrow 1 indicates the time of addition of CAP; arrow 2 indicates the time of addition of oxygen; arrow 3 indicates the time of removal of CAP. Each point is the mean of values for four plants
isomer of CAP, while effects on other processes are mediated by all the isomers (Ellis, 1963). Plants were exposed, therefore, to D-threo CAP or L-threo CAP, followed by 90% oxygen for 100 rain. It is seen (Fig. 3) that on the addition of 02 there is a rapid and complete inhibition of nitrogenase activity, as noted previously (Fig. 2). On removal of Oa there is a significant difference between the rates at which nitrogenase activity recovers from oxygen inactivation in the presence of the two isomers, with the L-threo plants being no different from the untreated control whereas the D-threo plants recover more slowly and to a much lesser extent. These results suggest that the main effect of D-threo CAP in these studies is on protein synthesis. The inhibitory effects of both isomers on nitrogenase activity are reversible with acetylene reduction increasing when the inhibitors are removed. This implies that the more rapid recovery of nitrogenase from oxygen in the presence of L-threo CAP was not due to a selective breakdown of the L-threo isomer within the plant.
4. The Effect of Cycloheximide Concentration on Nitrogenase Resynthesis In Fig. 2 it was shown that CH has no long-term effect on nitrogenase resynthesis and while this was possibly because the nitrogenase was synthesised on 70 S ribosomes, which would be unaffected by CH, it was also possible that the inhibitor was not penetrating the plant, or was not reaching a sufficiently high concentration to inhibit protein biosynthesis on 80 S ribosomes. An experiment was therefore carried out
4
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase 1
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recovers when the inhibitor is removed. When oxygen is added in the absence of inhibitor (Fig. 4b) nitrogenase activity is inhibited almost immediately, but as in Figs. 2 and 3, recovers quickly to its initial level on removal of oxygen. When CH is added, followed by oxygen (Fig. 4c) there is an initial decline due to CH, followed by an immediate inhibition by oxygen. On removal of oxygen, activity recovers initially, as in the plants without CH, but then drops off. The initial and rapid recovery of nitrogenase activity in CH-treated plants after removal of oxygen provides evidence that the effect of CH is not directly on the synthesis of nitrogenase and suggests either that the synthesis of some plant-synthesised protein which is necessary for nitrogenase activity is being inhibited, or that CH is affecting general plant metabolism. The fact that some recovery of nitrogenase activity occurred after removal of the CH from the plants indicates the reversible nature of this inhibition.
1
5. The Effects of CAP and CH on Protein Biosynthesis and Nitrogenase Activity
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Time (days)
Fig. 4a-c. The effects of CH and oxygen on nitrogenase activity by intact plants of Alnus glutinosa. (a) The effect of the addition of 67 ~tM CH and its subsequent removal on nitrogenase activity by intact plants of Alnus glutinosa. (b) The effect of oxygen (90% for 100 min) on nitrogenase activity of plants not previously treated with CH. (c) The effect of oxygen (90% for 100 min) on nitrogenase activity of plants previously treated with 67 p.M CH and the effect of subsequent removal of the CH. Arrow 1 indicates the time of addition of CH; arrow 2 indicates the time of removal of CH; arrow 3 indicates the time of addition of oxygen. In experiment 4c there was no significant difference (P=0.05 level) between the activities of plants of the first 7 samplings (untreated plants). The activities of plants at samplings 9 onwards were significantly lower than the untreated controls and at samplings 10-12 were significantly higher than those at samplings 9-13. Activity at the last sampling was significantly higher than at samplings 13-15
in which the concentration of CH was increased to 67 gM, a concentration much in excess of that normally used in studies with this inhibitor (Macdonald and Ellis, 1969). The results show that when CH is added in the absence of oxygen (Fig. 4a), nitrogenase activity is inhibited completely within 3 days, but
The above results suggest that nitrogenase resynthesis is inhibited directly by CAP but not by CH and indicates, therefore, that nitrogenase is made on 70 S ribosomes. Before this view can be accepted with certainty, it is necessary to show two additional things : first, that the concentration of CAP which inhibits nitrogenase resynthesis has no effect on protein synthesis by the higher plant; and second, that the concentration of CH which has no effect on nitrogenase resynthesis inhibits protein synthesis by the higher plant. Experiments were carried out, therefore, in which protein biosynthesis by the higher plant roots and nodules was followed in the presence ofinhibitors during the time that nitrogenase was being resynthesised. To prevent problems due to possible contaminating microorganisms, the amino-acids in the roots and nodules were labelled by supplying the plant with 14CO2 which was fixed photosynthetically and then translocated to the roots and nodules, where it was incorporated into protein. Preliminary experiments showed that significant amounts of radioactivity arrived in the nodules of these plants within three hours of treatment with ~4CO2 and that the total photosynthate transferred from the leaves to the roots and nodules was the same irrespective of whether the plants were treated with CH, CAP, or neither. The procedure used in subsequent tests was as follows. Nitrogenase activity was monitored in a set of plants, in the usual way, over 7 days. The plants were then sub-divided into three groups of seven of similar size. One group was treated with 0.3 mM CAP, a second
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase Table 1. The effect of chloramphenicol and cydoheximide on nitrogenase activity and protein synthesis in Alnus Series
Controls Cycloheximide-treated Chloramphenicol-treated
Nitrogenase activity nmol C2U 4 (plant)-1 h-1
Protein synthesis nCi 14C incorporated into homogenate
Mean activity prior to 02 treatment
Mean activity 42 h after removing oxygen
Rate after removing 02 as % of initial activity
Roots
% activity Nodules after inhibitor treatment compared with the control
% activity after inhibitor treatment compared with the control
70 64 46
79 53 13
113 83 28
25 (9.3) 12 (5.1) 25 (7.8)
-
58 81
48 100
26 (4.7) 15 (7.6) 21 (3.7)
The plants were treated with inhibitor for 24 h prior to the addition of 90% 02 for 100 min. Nitrogenase activity was measured 42 h after the removal of 02. Each value is the mean of 7 replicates. Figures in brackets are standard deviations.
group was treated with 30/aM CH and the third group remained untreated. Twenty-four hours later, each set of plants was exposed to 90% 02 for 100 min, as previously described, followed immediately by exposure to 14CO2 for 42 hours. Protein was then extracted from each plant and its t4C-label determined. The results (Table 1) show, despite the high standard deviations which are inherent in an experiment of this type with whole Alnus plants, that in the series without added inhibitor, and in the presence of CH, there is a marked recovery in acetylene reduction after removal of the oxygen, whereas the CAP-treated plants recover only by 28 %. On the other hand, CH markedly inhibits protein synthesis both in the roots and in the nodules, thus showing that the CH penetrates these and is active within them. CAP has no effect on protein synthesis by the roots and inhibits total protein synthesis in the nodules only by 16%, possibly by specifically inhibiting protein biosynthesis on the prokaryotic (70 S) ribosomes. Thus, CH, at a concentration which inhibits eukaryotic protein biosynthesis, has no significant effect on the recovery of nitrogenase from inhibition by oxygen while CAP, at a concentration which inhibits recovery of nitrogenase activity from oxygen, has no significant effect on eukaryotic protein biosynthesis.
Discussion
To date there has been no evidence, apart from the strong analogy with legume root nodules, that the microbial symbiont (the actinomycete) within nonlegume root nodules is the site of synthesis of the nitrogenase enzyme. This paper provides experimental evidence in support of this widely held, but hitherto unproven, view.
The basis for this conclusion is three-fold. First, D-threo CAP, which inhibits protein synthesis on 70 S ribosomes, is a more effective inhibitor of nitrogenase activity than is CH, a specific inhibitor of 80 S ribosome activity, at concentrations where CH, but not CAP, inhibits protein synthesis by the roots. Erythromycin, another inhibitor of protein synthesis on 70 S ribosomes, is similar to CAP, and unlike CH, in inhibiting nitrogenase resynthesis in Alnus (data not shown). Second, the major source of 70 S ribosomes in the root nodules is the endophyte. Third, although it is possible to hypothesise that the effect of D-threo CAP may not be specifically on the synthesis of the nitrogenase protein, and that CAP may be inhibiting the synthesis of some protein essential for nitrogenase activity, for example one involved in reductant supply, rather than nitrogenaseper se, this seems unlikely. For example, this protein would have to be oxygen-labile and it is more logical to assume that the inhibitory effect of CAP is directly on the synthesis of nitrogenase, a known oxygen-labile protein, rather than to implicate some other hypothetical oxygen-labile protein. The argument that the nitrogenase is perhaps being made on mitochondrial 70 S ribosomes of the host rather than on 70 S ribosomes of the endophyte is unlikely, since there is no analogy for this anywhere in the plant kingdom. An additional point relating to nitrogenase synthesis within non-legume root nodules is the question of whether the enzyme is genetically coded for by the endophyte or by the host. Until recently the evidence that in the Rhizobium-legume symbiosis the nitrogenase was coded for by the prokaryote was suggestive, but equivocal (see Phillips et al., 1973; Dunican and Tierney, 1974). There is now evidence based on the ability of certain rhizobia to fix N2 in the absence of the host (Kurz and LaRue, 1975; McComb et al., 1975; Pagan et al., 1975) that the nitrogenase is also
6
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
genetically coded for by the prokaryote. It is likely that this situation also applies in the non-legumeactinomycete symbiosis, but definite proof will be obtained only when the endophyte has been isolated and shown to fix N2 in axenic culture. This work was made possible through research support to W.D.P. Stewart from the Science Research Council, the Natural Environment Research Council and the Royal Society. R.A. Skeffington thanks the Science Research Council for a research studentship.
References Bond, G. : The fixation of nitrogen associated with the root nodules of Myrica gale L., with special reference to its pH relation and ecological significance. Ann. Bot. N.S. 15, 447-459 (1951) Bond, G. : Root nodule symbiosis with actinomycete-like organisms. In : The Biology of Nitrogen Fixation. pp. 342-378. Ed. : Quispel, A. Amsterdam: North Holland Publ./American Elsevier 1974 Boulter, D., Ellis, R.J., Yarwood, A. : Biochemistry of protein synthesis in plants. Biol. Rev. 47, 113-175 (1972) Dalton, H., Mortenson, L.E. : Dinitrogen fixation (with a biochemical emphasis). Bact. Rev. 36, 231-260 (1972) Dunican, L.K., Tierney, A.B. : Genetic transfer of nitrogen fixation from Rhizobium trifolii to Klebsiella aerogenes. Biochem. biophys. Res. Commun. 57, 62 71 (1974) Ellis, R.J. : Chloramphenicol and the uptake of salt in plants. Nature (Lond.) 200, 596-597 (1963) Ellis, R.J., Macdonald, I.R.: Activation of protein synthesis by microsomes from ageing beet discs. Plant Physiol. 42, 1297-1302 (1967)
Evans, H.J., Phillips, D.A. : Reductants for nitrogenase and relationships to cellular electron transport. In: Nitrogen Fixation by Free-Living Micro-Organisms. pp. 389-419. Ed.: Stewart, W.D.P. Cambridge University Press 1975 Kennel, D. : Use of filters to separate radioactivity in RNA, DNA and protein. In: Methods in enzymology. Vol. 12, pp. 686-693. Eds.: Grossman, L., Moldave, E. New York: Academic Press 1967 Kurz, W.G.W., LaRue, T.A. : Nitrogenase activity in rhizobia in absence of plant host. Nature (Lond.) 256, 407-408 (1975) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin reagent. J. biol. Chem. 193, 265-275 (1951) Macdonald, I.R., Ellis, R.J. : Does cycloheximide inhibit protein synthesis specifically in plant tissues ? Nature (Lond.) 222, 791792 (1969) McComb, J.A., Elliott, J., Dilworth, M.J.: Acetylene reduction by Rhizobium in pure culture. Nature (Lond.) 256, 409-410 (1975) Pagan, J.D., Child, J.J., Scowcroft, W.R., Gibson, A.H. : Nitrogen fixation by Rhizobium cultured on a defined medium. Nature (Lond.) 256, 406-407 (1975) Patterson, M.S., Greene, R.C. : Measurement of low energy fl-emitters in aqueous solution by liquid scintillation counting of emulsions. An. Chem. 37, 854-857 (1965) Phillips, D.A., Howard, R.L., Evans, H.J.: Studies on the genetic control of a nitrogenase component in leguminous root nodules. Physiol. Plantarum (Kbh.) 28, 248-253 (1974) Stewart, W.D.P., Fitzgerald, G.P., Burris, R.H.: In situ studies on nitrogen fixation using the acetylene reduction technique. Proc. nat. Acad. Sci. (Wash.) 58, 2071-2078 (1967)
Received 18 July; accepted 9 September 1975
Planta (Beri.) 129, 1 - 6 (I976)
9 by Springer-Verlag 1976
Evidence from Inhibitor Studies that the Endophyte Synthesises Nitrogenase in the Root Nodules of Alnus glutinosa L. Gaertn.* R.A. Skeffington** and W.D.P. Stewart Department of Biological Sciences, University of Dundee, Dundee, DD1 4 HN, U.K.
Summary. Nitrogenase activity (acetylene reduction assay) in the nodulated non-leguminous angiosperm Alnus glutinosa is inhibited within minutes when plants are exposed to a gas phase containing 90% oxygen. On returning the plants to air, nitrogenase activity recovers within a few hours, both in the presence of cycloheximide, which inhibits protein synthesis on 80 S (eukaryotic) ribosomes, and in the absence of inhibitor. When chloramphenicol, which inhibits protein synthesis on 70 S (prokaryotic) ribosomes, is added instead ofcycloheximide, recovery from oxygen inhibition does not occur, or occurs only slowly. The effects of chloramphenicol are specific to the D-threo-isomer which indicates a direct inhibition of protein synthesis. Erythromycin has a similar effect to chloramphenicol. Protein biosynthesis in non-nodulated roots is inhibited by cycloheximide but not by chloramphenicol. The results are interpreted as evidence that the nitrogenase within Alnus glutinosa root nodules is synthesised by the microbial symbiont.
Introduction
Over a hundred species of nodulated non-leguminous plants are known to fix N2 (Bond, 1974). They do so only in symbiosis with a micro-organism whose identity has never been established convincingly, but which is thought to be an actinomycete. In the legumeRhizobiurn symbiosis, it is known from nodule fractionation experiments (see e.g. Evans and Phillips, 1975) that the nitrogenase is located in the nodule bacteroids, and there is also recent evidence that it A b b r e v i a t i o n s . CAP = chloramphenicol; CH = cycloheximide. ** Present address : Department of Botany, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. *
is genetically coded for by the microbial symbiont, even if this information is usually repressed in the free-living state (see Phillips et al., 1973; Dunican and Tierney, 1974; Kurz and LaRue, t975; McComb et al., 1975; Pagan et aL, 1975). However, it is not possible to locate the site of nitrogenase activity in nonlegume root nodules using fractionation experiments, since consistently high nitrogen-fixing preparations of such nodules have not yet been obtained, neither has the microbial symbiont been isolated with certainty in the free-living state, let alone shown to fix nitrogen. In an attempt to decide whether the nitrogenase in the root nodules of Alnus glutinosa is synthesised by the prokaryotic or eukaryotic symbiont, we have used a different approach to those already tried. That is, we have inhibited the nitrogenase in intact root nodules irreversibly by adding high levels of oxygen (see Dalton and Mortenson, 1972) and then followed nitrogenase resynthesis in the presence of inhibitors of protein biosynthesis specific to either prokaryotes or eukaryotes. The inhibitors used were chloramphenicol (CAP) which inhibits protein biosynthesis on prokaryotic type (70 S) ribosomes only and cycloheximide (CH) which inhibits this activity on eukaryotic type (80 S) ribosomes only (see Boulter et aL, 1972). In the studies using CAP, both the D-threo and L-threo isomers exert various inhibitory effects on general cell metabolism, but the D-threo isomer is the only one which, in addition, exerts a specific effect on protein biosynthesis (Ellis, 1963). This provides a method of distinguishing whether an observed effect of CAP is due to an inhibition of protein biosynthesis or not. In the case of CH, it has also to be shown that any in vivo effect is specifically on protein biosynthesis and not on some other metabolic process (see e.g. Ellis, 1963; Macdonald and Ellis, 1969). In addition, any effect on protein biosynthesis must be demonstrated directly.
2
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
Materials and Methods Culture of Plants. Seed of Alnus glutinosa L. Gaertn. was collected in the east of Scotland, air dried, and stored in Petri dishes at a temperature of 4 ~ C and a relative humidity of 40%. The seed was germinated when required on moist sterile filter paper in Petri dishes after first being surface sterilized by immersion in 10% sodium hypochlorite for 15 min followed by several washes in sterile distilled water. The dishes were kept in the light at 20 ~ C. About 14% of the seeds germinated_ After ten days, the seedlings were transferred to polythene trays containing one quarter-strength nitrogen-free Crone's solution (Bond, 1951) solidified with 0.8 % (w/v) agar. These were maintained in growth cabinets under a 16 h:8 h light-dark regime, with a day temperature of 22 ~ C, a night temperature of 18~ C and an illuminance of 7,000 lux during the light period. Once the first true leaf appeared, the root system of each plant was treated with 0.5 ml of a suspension of crushed Alnus root nodules, which contained approximately 10 g fresh weight of nodules per 100 ml of distilled water. Nodules appeared on all plants within two weeks, and after a further 2 4 weeks growth in the agar trays, the plants were used for experimentation. Preparation of Plants for Experiments. Uniform batches of plants, aged 6-8 weeks, were selected for each experiment. Each plant was placed in a sterilised test-tube containing 2.5 ml of nitrogen-free full strength Crone's solution. The plants were supported by lightly tying the stem to a polythene strip placed in the tube. The bottom of each tube around the roots was painted black and the top closed With cotton wool, except during the acetylene reduction assays (see below) when a serum stopper replaced the cotton wool. The plants were subjected to the various inhibitor treatments 7 days after transfer to the test-tubes. Treatment of Plants with Inhibitors and Oxygen. D-threo chloramphenicol, erythromycin and cycloheximide were purchased from Sigma Ltd., London: L-threo chloramphenicol was a gift from Dr. R.E. Bowman, Parke-Davis Ltd., Pontypool. The inhibitors were dissolved freshly in Crone's solution before use. At the start of the differential treatments, plants were selected at random and their culture solution replaced by the inhibitor solution. All solutions, including those in the control tubes, were renewed every 3 days. Plants were treated with oxygen by evacuating the sealed test-tubes containing the plants and then adding 90% 02 in air to atmospheric pressure. After exposure of the plants to this gas phase for 100 min, air was re-admitted and assays for acetylene reduction were carried out at intervals thereafter. Acetylene Reduction Assays. These were carried out in each test-tube, under a gas phase of 12% (v/v) acetylene in air. Acetylene reduction was followed in 4 plants for each treatment over the whole experimental period. After a 30 min incubation period in the presence of acetylene, a sample of the gas phase was withdrawn, without terminating the reaction, and assayed for acetylene reduction by gas chromatography (Stewart et al., 1967). At the end of the experiment, the nodules were excised from the plants and a check for free-living nitrogen-fixing contaminants (which were never found) was carried out by assaying the remainder of each plant for a capacity to reduce acetylene to ethylene. The Measurement of Protein Biosynthesis in Roots and Nodules. In these experiments, the plants were treated with inhibitor (0.3 mM CAP and 30 gM CH) followed 24 h later by 90% 02 for 100 rain to inactivate the nitrogenase. Immediately after the oxygen treatment, each intact plant was given 26 gl of 1.66 mM NaH14COs (Radiochemical Centre, Amersham), containing a total of 2.6 pCi 14C, for a period of 42 h. The pH of the culture solution did not change significantly from 5.5 during this treatment. The protein was then extracted separately from the roots and from the nodules (Kennell, 1967) and the total dry weight of extracted matter was also determined. An aliquot of the final solu-
tion was taken for protein determination (Lowry et al., 1951) after sotubilisation of the precipitate with 1 N NaOH. The mean protein content of the precipitates ranged from 80 105%. Radioactivity incorporated into each sample was measured by scintillation counting using the scintillant of Patterson and Greene (1965) and a Tracerlab Corumatic 200 liquid scintillation counter. Quenching was corrected for by the channels ratio method using aniline as standard quenching agent. The counting efficiency was approximately 40%.
Results
1. The Effects of Chloramphenicol and Cycloheximide on Nitrogenase Activity The effects of CAP and CH on nitrogenase activity were checked by adding concentrations of inhibitors reported previously to inhibit prokaryotic (0.5 mM CAP) and eukaryotic (36 pM CH) protein synthesis (Ellis and Macdonald, 1967; Macdonald and Ellis, 1969). The results (Fig. 1) show that CAP abolishes nitrogenase activity almost completely and irreversibly within 48 h. With CH, on the other hand, there is an initial inhibition of acetylene reduction, possibly due to an effect on general cell metabolism, but activity then recovers fairly quickly to its original level. These results show that CAP, the inhibitor of protein synthesis in prokaryotes, is a more effective and persistent inhibitor of nitrogenase activity in intact nodules than is CH, the inhibitor of protein synthesis in eukaryotes.
2. The Effects of Chloramphenicol and Cycloheximide on Recovery of Nitrogenase Activity from Oxygen Inhibition In this experiment, the inhibitors were added, followed by 90% 02 for 100 min to specifically inactivate the
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R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
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nitrogenase, and then recovery of nitrogenase activity was monitored after removal of 02. The results (Fig. 2) show that there is some decline in nitrogenase activity when the inhibitors are added, although the differences between the three treatments (CAP, CH, and untreated control) are not significant at the P--0.05 level. When oxygen is added, nitrogenase activity is almost totally abolished within 5 min in all treatments. On removing the oxygen, nitrogenase activity in the untreated control and in the CH-treated series recovers fairly rapidly and there is no significant difference between these two treatments. However nitrogenase activity recovers more slowly in the CAP-treated plants and throughout the experimental period remains significantly lower than in the other two treatments. These data show that when nitrogenase is inhibited specifically by oxygen and the oxygen then removed, nitrogenase activity recovers rapidly in the presence of the inhibitor of eukaryotic protein synthesis (CH), but does not recover to the same extent in the presence of CAP which inhibits prokaryotic protein synthesis.
3. The Effects of Different Optical Isomers of Chloramphenicol on Nitrogenase Activity These experiments provide evidence that the inhibitory effects of CAP on nitrogenase activity noted in Fig. 1 and Fig. 2 are probably due to an inhibition of protein synthesis. As discussed earlier, protein synthesis in prokaryotes is inhibited only by the D-threo
Fig. 3. The effects of oxygen (90% for 100 rain) on nitrogenase activity by intact plants of Alnus glutinosa treated with 0.3 mM Dthreo CAP ( e - e ) and 0.3 mM L-threo CAP (=-,,), or untreated (A-A). From the point of removal of oxygen, the recovery of D-threo CAP-treated plants differs significantly from the other two treatments (P=0.05 level). The latter treatments do not differ significantly form each other except at the last sampling time. Arrow 1 indicates the time of addition of CAP; arrow 2 indicates the time of addition of oxygen; arrow 3 indicates the time of removal of CAP. Each point is the mean of values for four plants
isomer of CAP, while effects on other processes are mediated by all the isomers (Ellis, 1963). Plants were exposed, therefore, to D-threo CAP or L-threo CAP, followed by 90% oxygen for 100 rain. It is seen (Fig. 3) that on the addition of 02 there is a rapid and complete inhibition of nitrogenase activity, as noted previously (Fig. 2). On removal of Oa there is a significant difference between the rates at which nitrogenase activity recovers from oxygen inactivation in the presence of the two isomers, with the L-threo plants being no different from the untreated control whereas the D-threo plants recover more slowly and to a much lesser extent. These results suggest that the main effect of D-threo CAP in these studies is on protein synthesis. The inhibitory effects of both isomers on nitrogenase activity are reversible with acetylene reduction increasing when the inhibitors are removed. This implies that the more rapid recovery of nitrogenase from oxygen in the presence of L-threo CAP was not due to a selective breakdown of the L-threo isomer within the plant.
4. The Effect of Cycloheximide Concentration on Nitrogenase Resynthesis In Fig. 2 it was shown that CH has no long-term effect on nitrogenase resynthesis and while this was possibly because the nitrogenase was synthesised on 70 S ribosomes, which would be unaffected by CH, it was also possible that the inhibitor was not penetrating the plant, or was not reaching a sufficiently high concentration to inhibit protein biosynthesis on 80 S ribosomes. An experiment was therefore carried out
4
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase 1
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recovers when the inhibitor is removed. When oxygen is added in the absence of inhibitor (Fig. 4b) nitrogenase activity is inhibited almost immediately, but as in Figs. 2 and 3, recovers quickly to its initial level on removal of oxygen. When CH is added, followed by oxygen (Fig. 4c) there is an initial decline due to CH, followed by an immediate inhibition by oxygen. On removal of oxygen, activity recovers initially, as in the plants without CH, but then drops off. The initial and rapid recovery of nitrogenase activity in CH-treated plants after removal of oxygen provides evidence that the effect of CH is not directly on the synthesis of nitrogenase and suggests either that the synthesis of some plant-synthesised protein which is necessary for nitrogenase activity is being inhibited, or that CH is affecting general plant metabolism. The fact that some recovery of nitrogenase activity occurred after removal of the CH from the plants indicates the reversible nature of this inhibition.
1
5. The Effects of CAP and CH on Protein Biosynthesis and Nitrogenase Activity
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Fig. 4a-c. The effects of CH and oxygen on nitrogenase activity by intact plants of Alnus glutinosa. (a) The effect of the addition of 67 ~tM CH and its subsequent removal on nitrogenase activity by intact plants of Alnus glutinosa. (b) The effect of oxygen (90% for 100 min) on nitrogenase activity of plants not previously treated with CH. (c) The effect of oxygen (90% for 100 min) on nitrogenase activity of plants previously treated with 67 p.M CH and the effect of subsequent removal of the CH. Arrow 1 indicates the time of addition of CH; arrow 2 indicates the time of removal of CH; arrow 3 indicates the time of addition of oxygen. In experiment 4c there was no significant difference (P=0.05 level) between the activities of plants of the first 7 samplings (untreated plants). The activities of plants at samplings 9 onwards were significantly lower than the untreated controls and at samplings 10-12 were significantly higher than those at samplings 9-13. Activity at the last sampling was significantly higher than at samplings 13-15
in which the concentration of CH was increased to 67 gM, a concentration much in excess of that normally used in studies with this inhibitor (Macdonald and Ellis, 1969). The results show that when CH is added in the absence of oxygen (Fig. 4a), nitrogenase activity is inhibited completely within 3 days, but
The above results suggest that nitrogenase resynthesis is inhibited directly by CAP but not by CH and indicates, therefore, that nitrogenase is made on 70 S ribosomes. Before this view can be accepted with certainty, it is necessary to show two additional things : first, that the concentration of CAP which inhibits nitrogenase resynthesis has no effect on protein synthesis by the higher plant; and second, that the concentration of CH which has no effect on nitrogenase resynthesis inhibits protein synthesis by the higher plant. Experiments were carried out, therefore, in which protein biosynthesis by the higher plant roots and nodules was followed in the presence ofinhibitors during the time that nitrogenase was being resynthesised. To prevent problems due to possible contaminating microorganisms, the amino-acids in the roots and nodules were labelled by supplying the plant with 14CO2 which was fixed photosynthetically and then translocated to the roots and nodules, where it was incorporated into protein. Preliminary experiments showed that significant amounts of radioactivity arrived in the nodules of these plants within three hours of treatment with ~4CO2 and that the total photosynthate transferred from the leaves to the roots and nodules was the same irrespective of whether the plants were treated with CH, CAP, or neither. The procedure used in subsequent tests was as follows. Nitrogenase activity was monitored in a set of plants, in the usual way, over 7 days. The plants were then sub-divided into three groups of seven of similar size. One group was treated with 0.3 mM CAP, a second
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase Table 1. The effect of chloramphenicol and cydoheximide on nitrogenase activity and protein synthesis in Alnus Series
Controls Cycloheximide-treated Chloramphenicol-treated
Nitrogenase activity nmol C2U 4 (plant)-1 h-1
Protein synthesis nCi 14C incorporated into homogenate
Mean activity prior to 02 treatment
Mean activity 42 h after removing oxygen
Rate after removing 02 as % of initial activity
Roots
% activity Nodules after inhibitor treatment compared with the control
% activity after inhibitor treatment compared with the control
70 64 46
79 53 13
113 83 28
25 (9.3) 12 (5.1) 25 (7.8)
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58 81
48 100
26 (4.7) 15 (7.6) 21 (3.7)
The plants were treated with inhibitor for 24 h prior to the addition of 90% 02 for 100 min. Nitrogenase activity was measured 42 h after the removal of 02. Each value is the mean of 7 replicates. Figures in brackets are standard deviations.
group was treated with 30/aM CH and the third group remained untreated. Twenty-four hours later, each set of plants was exposed to 90% 02 for 100 min, as previously described, followed immediately by exposure to 14CO2 for 42 hours. Protein was then extracted from each plant and its t4C-label determined. The results (Table 1) show, despite the high standard deviations which are inherent in an experiment of this type with whole Alnus plants, that in the series without added inhibitor, and in the presence of CH, there is a marked recovery in acetylene reduction after removal of the oxygen, whereas the CAP-treated plants recover only by 28 %. On the other hand, CH markedly inhibits protein synthesis both in the roots and in the nodules, thus showing that the CH penetrates these and is active within them. CAP has no effect on protein synthesis by the roots and inhibits total protein synthesis in the nodules only by 16%, possibly by specifically inhibiting protein biosynthesis on the prokaryotic (70 S) ribosomes. Thus, CH, at a concentration which inhibits eukaryotic protein biosynthesis, has no significant effect on the recovery of nitrogenase from inhibition by oxygen while CAP, at a concentration which inhibits recovery of nitrogenase activity from oxygen, has no significant effect on eukaryotic protein biosynthesis.
Discussion
To date there has been no evidence, apart from the strong analogy with legume root nodules, that the microbial symbiont (the actinomycete) within nonlegume root nodules is the site of synthesis of the nitrogenase enzyme. This paper provides experimental evidence in support of this widely held, but hitherto unproven, view.
The basis for this conclusion is three-fold. First, D-threo CAP, which inhibits protein synthesis on 70 S ribosomes, is a more effective inhibitor of nitrogenase activity than is CH, a specific inhibitor of 80 S ribosome activity, at concentrations where CH, but not CAP, inhibits protein synthesis by the roots. Erythromycin, another inhibitor of protein synthesis on 70 S ribosomes, is similar to CAP, and unlike CH, in inhibiting nitrogenase resynthesis in Alnus (data not shown). Second, the major source of 70 S ribosomes in the root nodules is the endophyte. Third, although it is possible to hypothesise that the effect of D-threo CAP may not be specifically on the synthesis of the nitrogenase protein, and that CAP may be inhibiting the synthesis of some protein essential for nitrogenase activity, for example one involved in reductant supply, rather than nitrogenaseper se, this seems unlikely. For example, this protein would have to be oxygen-labile and it is more logical to assume that the inhibitory effect of CAP is directly on the synthesis of nitrogenase, a known oxygen-labile protein, rather than to implicate some other hypothetical oxygen-labile protein. The argument that the nitrogenase is perhaps being made on mitochondrial 70 S ribosomes of the host rather than on 70 S ribosomes of the endophyte is unlikely, since there is no analogy for this anywhere in the plant kingdom. An additional point relating to nitrogenase synthesis within non-legume root nodules is the question of whether the enzyme is genetically coded for by the endophyte or by the host. Until recently the evidence that in the Rhizobium-legume symbiosis the nitrogenase was coded for by the prokaryote was suggestive, but equivocal (see Phillips et al., 1973; Dunican and Tierney, 1974). There is now evidence based on the ability of certain rhizobia to fix N2 in the absence of the host (Kurz and LaRue, 1975; McComb et al., 1975; Pagan et al., 1975) that the nitrogenase is also
6
R.A. Skeffington and W.D.P. Stewart: Endophyte Synthesises Nitrogenase
genetically coded for by the prokaryote. It is likely that this situation also applies in the non-legumeactinomycete symbiosis, but definite proof will be obtained only when the endophyte has been isolated and shown to fix N2 in axenic culture. This work was made possible through research support to W.D.P. Stewart from the Science Research Council, the Natural Environment Research Council and the Royal Society. R.A. Skeffington thanks the Science Research Council for a research studentship.
References Bond, G. : The fixation of nitrogen associated with the root nodules of Myrica gale L., with special reference to its pH relation and ecological significance. Ann. Bot. N.S. 15, 447-459 (1951) Bond, G. : Root nodule symbiosis with actinomycete-like organisms. In : The Biology of Nitrogen Fixation. pp. 342-378. Ed. : Quispel, A. Amsterdam: North Holland Publ./American Elsevier 1974 Boulter, D., Ellis, R.J., Yarwood, A. : Biochemistry of protein synthesis in plants. Biol. Rev. 47, 113-175 (1972) Dalton, H., Mortenson, L.E. : Dinitrogen fixation (with a biochemical emphasis). Bact. Rev. 36, 231-260 (1972) Dunican, L.K., Tierney, A.B. : Genetic transfer of nitrogen fixation from Rhizobium trifolii to Klebsiella aerogenes. Biochem. biophys. Res. Commun. 57, 62 71 (1974) Ellis, R.J. : Chloramphenicol and the uptake of salt in plants. Nature (Lond.) 200, 596-597 (1963) Ellis, R.J., Macdonald, I.R.: Activation of protein synthesis by microsomes from ageing beet discs. Plant Physiol. 42, 1297-1302 (1967)
Evans, H.J., Phillips, D.A. : Reductants for nitrogenase and relationships to cellular electron transport. In: Nitrogen Fixation by Free-Living Micro-Organisms. pp. 389-419. Ed.: Stewart, W.D.P. Cambridge University Press 1975 Kennel, D. : Use of filters to separate radioactivity in RNA, DNA and protein. In: Methods in enzymology. Vol. 12, pp. 686-693. Eds.: Grossman, L., Moldave, E. New York: Academic Press 1967 Kurz, W.G.W., LaRue, T.A. : Nitrogenase activity in rhizobia in absence of plant host. Nature (Lond.) 256, 407-408 (1975) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin reagent. J. biol. Chem. 193, 265-275 (1951) Macdonald, I.R., Ellis, R.J. : Does cycloheximide inhibit protein synthesis specifically in plant tissues ? Nature (Lond.) 222, 791792 (1969) McComb, J.A., Elliott, J., Dilworth, M.J.: Acetylene reduction by Rhizobium in pure culture. Nature (Lond.) 256, 409-410 (1975) Pagan, J.D., Child, J.J., Scowcroft, W.R., Gibson, A.H. : Nitrogen fixation by Rhizobium cultured on a defined medium. Nature (Lond.) 256, 406-407 (1975) Patterson, M.S., Greene, R.C. : Measurement of low energy fl-emitters in aqueous solution by liquid scintillation counting of emulsions. An. Chem. 37, 854-857 (1965) Phillips, D.A., Howard, R.L., Evans, H.J.: Studies on the genetic control of a nitrogenase component in leguminous root nodules. Physiol. Plantarum (Kbh.) 28, 248-253 (1974) Stewart, W.D.P., Fitzgerald, G.P., Burris, R.H.: In situ studies on nitrogen fixation using the acetylene reduction technique. Proc. nat. Acad. Sci. (Wash.) 58, 2071-2078 (1967)
Received 18 July; accepted 9 September 1975
