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Effects of host on the expression of the H2-uptake hydrogenase of Rhizobium in legume nodules
Plant Science Letters, 29 (1983) 191--199 Elsevier Scientific Publishers Ireland Ltd.
191
EFFECTS OF HOST ON THE EXPRESSION OF THE H2-UPTAKE HYDROGENASE OF RHIZOBIUM IN LEGUME NODULES
M. L6PEZ, V. C A R B O N E R O ,
E. C A B R E R A
and T. RUIZ-ARGi)ESO
Departamento de Microbiolog(a, E.T.S. de Ingenieros Agrdnomos, Madrid-3 (Spain) (Received June 15th, 1982) (Revision received September 24th, 1982) (Accepted September 24th, 1982)
SUMMARY
The relative efficiency of nitrogen fLxation by nodules and the H2-uptake hydrogenase activity of bacteroids produced by hydrogenase-positive strains of Rhizobium leguminosarum and R. japonicum in different legume hosts have been examined. Bacteroids from nodules of Pisum sativum and Vicia faba from strain 128C53 of R. legurninosarurn showed a capacity to take up H2, but no hydrogenase activity was detected in bacteroids from nodules of Lens culinaris inoculated with the same strain. Seven R. japonicum strains produced nodules in Glycine max and Vigna unguiculata and three of these were also able to nodulate Vigna radiata. In all these cases, the nodules evolved little or no H2 in air and the bacteroids isolated from strain 3Ilb6 had 10-fold less hydrogenase activity when isolated from V. radiata than when obtained from nodules of G. max or V. unguiculata. These results demonstrate a strain-dependent host effect on the expression of H2-uptake hydrogenase in legume nodules.
Key words: N i t r o g e n f i x a t i o n - - H 2 - u p t a k e h y d r o g e n a s e - - Rhizobium leguminosarum
INTRODUC~ON
Nodules from most agriculturally important legumes waste considerable amounts of energy in the production of hydrogen catalyzed by the nitrogen fixing system [1--5]. However, certain strains of Rhizobium japonicum [2,5--7], R. leguminosarum [8--10] and Rhizobium sp [2,6] have been Abbreviations: PMS, phenazine methosulfate.
0304-4211/83/0000--0000/$03.00 © Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
192 found to express in legume nodules a H2-uptake hydrogenase which is involved in recycling the H2 produced during the nitrogen fixation process. The oxidation of H2 bacteroids from soybean nodules produced by hydrogenase-positive (Hup +) strains of R. japonicum generates energy, supports nitrogenase activity and conserves endogenous substrates [ 8,11,12 ]. These findings with bacteroids and results obtained in experiments which compare yields and nitrogen content of soybean plants inoculated with Hup ÷ and Hup- strains of R. japonicum, provide support for the idea that the H~-uptake phenotype has a beneficial role in the legume symbiosis [ 13,14]. Hydrogenase activity is not expresed by Rhizobium in normal culture conditions, but the genes for the H2-uptake system are derepressed in nodule bacteroids and in free-living cultures under defined conditions [15]. Factors like carbon substrates, oxygen concentration and induction by H2, regulate the expression of hydrogenase in free living R. japonicum [ 16,17]. In the symbiotic state, nodule environment is supposed to trigger the derepression of the hydrogen-uptake genes. However, host factors affecting or controlling the induction and activity of the hydrogenase have not been described. Dixon [ 18] found different levels of hydrogenase activity in nodules produced by a strain of R. leguminosarum in different plant species of the same rhizobial cross-inoculation group but Carter et al. [5] did not observe differences in H2 evolution in nodules produced by a Hup + strain of R. japonicum in seven soybean cultivars. Here, we report a strain dependent host effect on the energy efficiency of nitrogen fixation and the expression of hydrogenase in nodules produced by Hup ÷ strains of R. leguminosarum and R. japonicum. MATERIALS AND METHODS
Cultures of Rhizobium Strain of R. leguminosarum 128C53 was received from Dr. J. Burton, Nitragin Co., MO, U.S.A.; strains of R. japonicum 3 I l b l l 0 , USDA122DES, 3Ilb142, 3Ilb143, USDA136 and 3 I l b 6 were obtained from Dr. H.J. Evans, Oregon State University, Corvallis, U.S.A.; USDA138 from Dr. H. Keyser, USDA, Beltsville, U.S.A.; 3.15B3 from the Facultd des Sciences Agronomics, Gembloux, Belgium; NU150 from Dr. J.M. Vincent, University of Sidney, Sidney, Australia; CB1003 from Dr. R.J. Roughley, Horticulture Research Station, Gosford, Australia. Cultures were maintained in yeast extract-mannitol slants [19] and stored at 4 ° C. Plant material and growth conditions Seeds of soybean (G. max L. cv. Amsoy), peas (P. sativum L. cv. Lincoln), blackeyed cowpeas (V. unguiculata L. Walp sup. unguiculata), green gram (V. radiata L. Wilczek), lentils (L. culinaris Medik.) and beans (V. faba L.) were surface disinfected [19] and germinated in 0.5% agar plates. After
193 3 days the germinated seeds were planted in autoclaved Leonard jars containing free-nitrogen plant nutrient solution [19]. Four seedlings per jar were inoculated with 10 ml of yeast extract-mannitol liquid culture of the appropriate Rhizobium strains. Plants were grown in a controlled environment chamber with a photoperiod of 15 h and irradiated with fluorescent and incandescent light at an intensity of 30 klux. The temperature during the day/night cycle was 27°/18°C for soybeans, cowpeas and green gram and 23°/15°C for peas, beans and lentils.
Assays of nodules Plants were harvested after 30 days of growth and nodules attached to segments of roots (2--5 cm) were excised. Acetylene reduction and H2 evolution rates were chromatographically determined in separate samples of nodules (0.2--0.4 g). Ethylene was measured as in Ref. 7 and H2 as described by Hanus et al. [ 20 ]. • Preparation and assay of bacteroids Bacteroids from each Rhizobium-legume combination were obtained from nodules collected from 5--10 Leonard jar culture units. Nodules were crushed and bacteroids prepared as described by Emerich et al. [11] except that anaerobic conditions were not maintained and final bacteroid pellet was suspended in 10 ml of Mg2+-phosphate buffer (50 mM potassium phosphate, 2.5 mM magnesium chloride, pH 7.0). Hydrogenase activity of the bacteroid suspensions was determined from the H~-uptake rates when H2 and an electron acceptor were externally supplied. Hydrogen and O~ in the aqueous phase were measured continuously and simultaneously by an amperometric technique [21]. Phenazine methosulfate (PMS) (2 mM) and methylene blue (1 mM) were used as artificial electron acceptors. Protein determinations The protein content of bacteroid suspensions were determined by digesting the cells according to Stickland [22] and using the micro-biuret method as described by Goa [23]. Bovin serum albumin was used as standard. Chemicals Hydrogen and O2 were purchased from Sociedad Espafiola de Oxygeno (Spain), C2H2 was generated from calcium carbide, PMS was obtained from Serva Feinbiochemica, F.R.G. and methylene blue from Merck AG, F.R.G. RESULTS In order to study the effect of the host on the expression of H2-oxidizing hydrogenases in legume nodules two characteristics are required in the Rhizobium strains to be used: (a) to possess the H2-uptake hydrogenase genes and (b) to be able to nodulate different host legumes.
194 Strain 128C53 of R. leguminosarum [9,10] and several strains of R. japonicurn [ 5, 7 ] have been reported to induce H2-uptake hydrogenases in peas and soybeans, respectively. In preliminary assays we tested the capacity of these strains to nodulate different legume species of the pea and cowpea rhizobial cross inoculation groups. Strain 128C53 was found to nodulate effectively lentils, beans and peas. All ten R. japonicum strains tested were able to effectively nodulate cowpeas, besides soybeans and six of them nodulated also green gram. Table I shows the relative efficiency of nitrogen fixation of nodules produced by strain 128C53 in peas, beans and lentils. Nodules from the three hosts evolved H2 in air. However when the rates of H2 evolution were compared with the C2H2 reduction rates as a measurement of the total nitrogenase activity, it was shown that while lentil nodules were loosing 49% o f the total energy available for nitrogenase, nodules of peas and beans were more efficient, losing only 17% and 36%, respectively. Bacteroids were extracted from the nodules and tested for hydrogenase activity with O2 and t w o non-physiological electron acceptors. The uptake of H2 externally supplied was followed amperometrically and Fig. 1 shows a typical recording obtained with bacteroids from pea nodules. Bacteroids from pea and bean nodules exhibited a capacity to consume H2 (Fig. 1 and Table I), indicating the presence of a H2-uptake system. However no detectable hydrogenase activity was found in bacteroids from lentil nodules, either with 02, PMS or methylene blue as final electron acceptors. Table II shows the relative efficiency of nodules and hydrogenase activities of bacteroids from eight H u p ÷ and t w o H u p - strains of R. japonicum in different hosts. With one exception, all Hup ÷ strains which evolved little or no H2 in soybean nodules, also evolved little or no H2 in the
TABLE I EFFICIENCY O F N I T R O G E N FIXATION O F N O D U L E S A N D H Y D R O G E N A S E ACTIVITY OF BACTEROIDS PRODUCED BY STRAIN 128C53 OF R. LEGUMINOSAR UM IN PEAS, BEANS AND LENTILS Legume host
Peas Beans Lentils
H~ produced in aira (~mol/h/g fresh nod. wt.)
4.4 7.5 23.2
C2H 2 reduced a (umol/h/g fresh nod. wt.)
26.3 20.5 47.9
Relative efficiency l-H2 aira
Hydrogenase activity of bacteroids (electron acceptors) b
C~H2 red.
02
PMS
MB
0.83 ± 0.04 0.64 ± 0.06 0.51 ± 0.07
0.81 0.53 <0.05
0.62 0.33 <0.05
0.73 0.32 <0.05
a Values are means of determinations made in 5 replicated plant cultures (±S.E.M.). Plants were harvested and measurements made after 30 days. b ~mol H 2 consumed/htmg protein. MB, methylene blue.
195 I
!
!
m c o
...
20
7 MB
'0
t-
0
',_ ..............
0®
"0
"r"
0
|
1 Time
I
2
I
3
4
(minutes'~
Fig. 1. Amperometric recordings of H z ( ) a n d O 2 ( - - - - - - ) consumption by bacteroids (1.30 mg protein) from pea nodules of R. le~mino~rum strMn 1 2 8 C 5 3 . A t the times indicated, H 2 ( 2 7 / ~ M ) , O: (13/~M) and methylene blue (1 raM) were added to the electrode chamber (2.5 ml) as Mg2+-phospl'mte buffer solutions.
other hosts tested. Relative efficiencies in these hosts ranged between 0.96 and 1.0. The bacteroids extracted from the nodules produced by all these strains in the different hosts exhibited hydrogenase activities of the same order. The exception was strain 3Ilb6, which evolved H2 in green gram at a rate 16-fold higher than in cowpeas and soybeans. Although hydrogenase activity was detected in green gram bacteroids from this strain, its rate was only 0.30/~mol/h/mg protein, versus 3.94 and 3.81/zmol/h/mg protein in soybean and cowpea, respectively. The low relative efficiencies of nodules and the low hydrogenase activities of bacteroids from green gram inoculated with strain 3Ilb6 were confirmed in three additional experiments. The two Hup- strains of R. japonicum 3.15B3 and USDA138 produced nodules in soybeans, cowpeas and green gram while evolved large amounts of H2 in air. Bacteroids from nodules of the three hosts showed no capacity to take up H2 externally supplied. Relative efficiencies however, markedly differ among hosts, cowpeas relative efficiencies being the lowest.
196
T A B L E II GROWTH AND ENERGY EFFICIENCY OF NITROGEN FIXATION OF SOYBEAN C O W P E A A N D G R E E N G ~ A M I N O C U L A T E D WITH H Y D R O G E N A S E - P O S I T I V E A N D H Y D R O G E N A S E - N E G A T I V E S T R A I N S O F R. JAPONICUM Strains
Hosts
H 2 evolution C 2 H 2 reduction a Relative in aira (umol/h/g efficiency b (umol/h/g fresh nod. wt.) fresh nod. wt.)
H2-uptake by bacteroids c
Hydrogenase positive USDA122DES
Soybeans Cowpeas Green gram
0.0 0.0 0.0
18.9 24.62 18.1
1.00 + 0.0 1.00 -+ 0.0 1.00 -+ 0.0
5.28 4.87 4.18
3Ilb110
Soybeans Cowpeas G r e e n gram
0.0 0.0 0.0
30.2 19.2 6.8
"1.00 ~ 0.0 1.00 ± 0.0 1.00 -+ 0.0
4.67 4.65 5.20
3Ilb6
Soybeans Cowpeas G r e e n gram
0.39 0.41 6.58
20.92 18.66 20.25
0.98 -+ 0 . 0 1 3.94 0.99 -+ 0 . 0 0 5 3.81 0.69 ± 0 . 0 1 0.30
NU150
Soybeans Cowpeas G r e e n gram
1.10 0.10 0.00
27.3 18.69 20.4
0 . 9 6 + 0.02 0.99 -+ 0.03 1.00 +- 0.0
3.15 5.93 4.45
CB1003
Soybeans Cowpeas
0.60 0.00
30.9 17.35
0.98 ± 0.01 1.00 -+ 0.0
3.08 5.78
3Ilb142
Soybeans Cowpeas
0.1 0.0
18.4 12.30
0.99 -+ 0 . 0 4 1.00 -+ 0.0
3.96 4.63
3Ilb143
Soybeans Cowpeas
0.0 0.0
16.30 16.86
1.00 -+ 0.0 1.00 ~ 0.0
3.88 5.44
USDA136
Soybeans Cowpeas
0.0 0.0
19.2 16.25
1.00 ± 0.0 1.00 ± 0.0
4.25 5.20
44.0 11.4 31.0
0.65 -+ 0 . 0 3 0.49 ± 0.06 0.75 -+ 0.05
0.0 0.0 0.0
20.7 11.12 24.5
0.77 ± 0 . 0 6 0 . 3 6 ± 0.08 0 . 6 4 ± 0.05
0.0 0.0 0.0
Hydrogenase negative 3.15B3
Soybeans Cowpeas G r e e n gram
15.2 5.6 8.66
USDA138
Soybeans Cowpeas G r e e n gram
4.28 6.82 8.89
a Values are m e a n s o f d e t e r m i n a t i o n s m a d e in 4 r e p l i c a t e d p l a n t cultures. P l a n t s were
h a r v e s t e d a n d m e a s u r e m e n t s m a d e a f t e r 33 d a y s o f g r o w t h . Values r e p o r t e d as 0.0 were < 0 . 0 5 ~,mol o f H 2 p r o d u c e d / h / g fresh n o d u l e weight. b I"H~ ( a i r ) / C 2 H ~ r e d u c e d . Values are m e a n s ± S . E . M . c ~ m o l o f H2 e o n s u m e d / h / m g p r o t e i n . Values r e p o r t e d as 0.0 were < 0 . 0 5 ~ m o l o f H2 consumed/h/rag protein.
197 DISCUSSION
Results in Table I clearly show a marked effect on the legume host on the relative efficiency and hydrogenase activity of nodules produced by R. leguminosarum strain 128C53. This strain, which was previously shown to be Hup ÷ in pea nodules [9,10], exhibited a H2-uptake capacity in bacteroids from pea and bean nodules, but not in bacteroids from lentil nodules. Since no H2-uptake was observed either with PMS or methylene blue as artificial electron acceptors, the lack of hydrogenase activity in bacteroids from lentils is likely due to the absence of an active hydrogenase rather than to a defect on the electron transport chain to O:. By estimating deuterium disappearance, Dixon [18] also found that strain ONA 311 of R. leguminosarum induced a H2-uptake hydrogenase in nodules of P. sativum but not in nodules of V. faba. A clear host effect on the relative efficiency and on the expression of hydrogenase of R. japonicum in nodules was not observed in seven out of eight Hup ÷ strains tested. The seven strains expressed similar levels of hydrogenase activity in bacteroids from nodules of soybeans and cowpeas, and three of them also in bacteroids from green gram (Table II). The nodules from all hosts evolved little or no H~ in air. Cm~er et al. [5] also found no differences in the energy efficiency of nodules produced in seven cultivars of soybeans by the Hup ÷ strain 3Ilb143 of R. japonicum. By contrast, strain 3Ilb6 induced 10-fold higher hydrogenase activity in bacteroids from soybean and cowpea nodules than in bacteroids from nodules of green gram, which produced large amounts of H2 in air. While this work was in progress two preliminary reports appeared which indicate a similar effect of the host: Gibson et al. [23] communicated a host controlled development of hydrogenase in nodules formed by Rhizobium sp strains CB756 and 32H1, and Keyser et al. [24] reported significantly higher rates of H~ evolution in soybean nodules than in cowpea nodules produced in both hosts by two strains of R. japonicum. These results show that the legume host effects the expression of the H~-uptake hydrogenase of Rhizobium in nodules, but this effect is closely dependent on the Rhizobium strain. The mechanism of this effect is unknown and perhaps it will remain so until we understand the exact function of the H2-uptake hydrogenases in the nitrogen fixing process. In vitro experiments with bacteroids show that the oxidation of externally supplied H~ by the H2-uptake system provides energy for nitrogenase [8,11 ]. Since hydrogenase induction in free-living cells of R. japonicum is regulated by the presence of carbon substrates in the culture medium [16,17], one may speculate that hydrogenase activity and expression in legume nodules are controlled by the quantity and quality of photosynthates supplied by the plant host to the nodules. As can be seen in Tables I and II, hydrogenase activity of bacteroids correlates with the relative efficiency of nodules. This fact provides
198 a d d i t i o n a l s u p p o r t f o r t h e p r o p o s e d role f o r t h e H 2 - u p t a k e h y d r o g e n a s e in l e g u m e n o d u l e s o f recycling t h e H2 g e n e r a t e d b y t h e N2-fixing s y s t e m [ 2 , 2 5 ] . E n e r g y efficiencies o f n i t r o g e n f i x a t i o n m a y be a f f e c t e d b y f a c t o r s o t h e r t h a n t h e p r e s e n c e o f H 2 - u p t a k e h y d r o g e n a s e s since t h e t w o H u p strains o f R. japonicum e x a m i n e d have significantly d i f f e r e n t relative efficiencies in s o y b e a n s , c o w p e a s and green gram. ACKNOWLEDGEMENTS We are grateful t o Drs. J. B u r t o n , H.J. Evans, H. K e y s e r , R.J. R o u g h l e y and J.M. V i n c e n t f o r p r o d d i n g Rhizobium strains. This r e s e a r c h was s u p p o r t e d b y a G r a n t f r o m F u n d a c i S n R a m S n Areces (Spain). REFERENCES
1 R.O.D. Dixon, Ann. Bot. N.S., 31 (1967) 179. 2 K.R. Schubert and H.J. Evans, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1207. 3 T. Ruiz-Argiieso, R.J. Maier and H.J. Evans, Appl. Environ. Microbiol., 37 (1979) 582. 4 M.I. Minguez and T. Ruiz-Argiieso, Curr. Microbiol., 4 (1980) 169. 5 K.R. Carter, N.T. Jennings, F.J. Hanus and H.J. Evans, Can. J. Microbiol., 24 (1978) 307. 6 K.R. Schubert, J.A. Engelke, S.A. Rumell and H.J. Evans, Plant Physiol., 60 (1977) 651. 7 T. Ruiz-Argiieso, E. Cabrera and M. Barate, Arch. Microbiol., 128 (1981) 275. 8 R.O.D. Dixon, Arch. Microbiol., 62 (1968) 272. 9 T. Ruiz-Argiieso, J. Hanus and H.J. Evans, Arch. Microbiol., 116 (1978) 113. 10 G.J. Bethlenfavay and D.A. Phillips, Plant Physiol., 63 (1979) 816. 11 D.W. Emerich, T. Ruiz-Argiieso, T.M. Ching and H.J. Evans, J. Bacteriol., 137 (1979) 153. 12 R.E. McCrae, J. Hanus and H.J. Evans, Biochem. Biophys. Res. Commun., 80 (1978) 384. 13 S.L. Albretch, R.J. Maier, F.J. Hanus, S.A. Russell, D.W. Emerich and H.J. Evans, Science, 203 (1979) 1255. 14 R.M. Zablotowicz, S.A. Rumell and H.J. Evans, Agron. J., 72 (1980) 555. 15 R.J. Maier, N.E.R. Cambell, F.J. Hanns, F.B. Sympson, S.A. Russell and H.J. Evans, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 3258. 16 R.J. Maier, F.J. Hanus and H.J. Evans, J. Bacteriol., 137 (1979) 824. 17 S.T. Lira and K.T. Shanmugam, Biochim. Biophys. Acta, 584 (1979) 479. 18 R.O.D. Dixon, Arch. Microbiol., 85 (1972) 193. 19 J.M. Vincent, A manual for the practical study of root-nodule bacteria, International Biological Program, Blackwell, Oxford, 1970. 20 J. Hanus, K.R. Carter and H.J. Evans, Techniques for measurements of hydrogen evolution, in: A. San Pietro (Ed.), Methods in Enzymology, Vol. 69, Academic Press, London, 1980, p. 731. 21 R. Wang, F.P. Healy and J. Meyers, Plant Physiol., 48 (1971) 108. 22 C.H. Stlckland, J. Gen. Microbiol., 5 (1951) 698. 23 J. Goa, Scand. J. Clin. Lab. Invest., 5 (1953) 218. 24 A.H. Gibson, B.L. Dreyfus, R.L. Lawn, J.I. Sprent and G.L. Turner, Host and environmental factors affecting hydrogen evolution and uptake, in: A.H. Gibson and W.E. Newton (Eds.), Proceedings of the Fourth International Symposium on Nitrogen Fixation, Australian Academy of Science, Canberra, 1981, p. 373.
199 25 H. Keyser, P. van Berkum and D. Weber, Nitrogen activity and hydrogen evolution by Rhizobium japonicum in symbiosis with siratro and cowpea, in: A.H. Gibson and W.E. Newton (Eds.), Proceedings of the Fourth International Symposium on Nitrogen Fixation, Australian Academy of Science, Canberra, 1981, p. 374. 26 H.J. Evans, D.W. Emerich, T. Ruiz-Argiieso, S.L. Albretch, R.J. Maier, F. Sympson and S.A. Russell, Hydrogen metabolism in legume nodules and rhizobia: some recent developments, in: H.G. Schlegel and K. Schneider (Eds.), Hydrogenases: Their Catalytic Activity Structure and Function, Erich Goltze, GSttingen, 1978, p. 287.
191
EFFECTS OF HOST ON THE EXPRESSION OF THE H2-UPTAKE HYDROGENASE OF RHIZOBIUM IN LEGUME NODULES
M. L6PEZ, V. C A R B O N E R O ,
E. C A B R E R A
and T. RUIZ-ARGi)ESO
Departamento de Microbiolog(a, E.T.S. de Ingenieros Agrdnomos, Madrid-3 (Spain) (Received June 15th, 1982) (Revision received September 24th, 1982) (Accepted September 24th, 1982)
SUMMARY
The relative efficiency of nitrogen fLxation by nodules and the H2-uptake hydrogenase activity of bacteroids produced by hydrogenase-positive strains of Rhizobium leguminosarum and R. japonicum in different legume hosts have been examined. Bacteroids from nodules of Pisum sativum and Vicia faba from strain 128C53 of R. legurninosarurn showed a capacity to take up H2, but no hydrogenase activity was detected in bacteroids from nodules of Lens culinaris inoculated with the same strain. Seven R. japonicum strains produced nodules in Glycine max and Vigna unguiculata and three of these were also able to nodulate Vigna radiata. In all these cases, the nodules evolved little or no H2 in air and the bacteroids isolated from strain 3Ilb6 had 10-fold less hydrogenase activity when isolated from V. radiata than when obtained from nodules of G. max or V. unguiculata. These results demonstrate a strain-dependent host effect on the expression of H2-uptake hydrogenase in legume nodules.
Key words: N i t r o g e n f i x a t i o n - - H 2 - u p t a k e h y d r o g e n a s e - - Rhizobium leguminosarum
INTRODUC~ON
Nodules from most agriculturally important legumes waste considerable amounts of energy in the production of hydrogen catalyzed by the nitrogen fixing system [1--5]. However, certain strains of Rhizobium japonicum [2,5--7], R. leguminosarum [8--10] and Rhizobium sp [2,6] have been Abbreviations: PMS, phenazine methosulfate.
0304-4211/83/0000--0000/$03.00 © Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
192 found to express in legume nodules a H2-uptake hydrogenase which is involved in recycling the H2 produced during the nitrogen fixation process. The oxidation of H2 bacteroids from soybean nodules produced by hydrogenase-positive (Hup +) strains of R. japonicum generates energy, supports nitrogenase activity and conserves endogenous substrates [ 8,11,12 ]. These findings with bacteroids and results obtained in experiments which compare yields and nitrogen content of soybean plants inoculated with Hup ÷ and Hup- strains of R. japonicum, provide support for the idea that the H~-uptake phenotype has a beneficial role in the legume symbiosis [ 13,14]. Hydrogenase activity is not expresed by Rhizobium in normal culture conditions, but the genes for the H2-uptake system are derepressed in nodule bacteroids and in free-living cultures under defined conditions [15]. Factors like carbon substrates, oxygen concentration and induction by H2, regulate the expression of hydrogenase in free living R. japonicum [ 16,17]. In the symbiotic state, nodule environment is supposed to trigger the derepression of the hydrogen-uptake genes. However, host factors affecting or controlling the induction and activity of the hydrogenase have not been described. Dixon [ 18] found different levels of hydrogenase activity in nodules produced by a strain of R. leguminosarum in different plant species of the same rhizobial cross-inoculation group but Carter et al. [5] did not observe differences in H2 evolution in nodules produced by a Hup + strain of R. japonicum in seven soybean cultivars. Here, we report a strain dependent host effect on the energy efficiency of nitrogen fixation and the expression of hydrogenase in nodules produced by Hup ÷ strains of R. leguminosarum and R. japonicum. MATERIALS AND METHODS
Cultures of Rhizobium Strain of R. leguminosarum 128C53 was received from Dr. J. Burton, Nitragin Co., MO, U.S.A.; strains of R. japonicum 3 I l b l l 0 , USDA122DES, 3Ilb142, 3Ilb143, USDA136 and 3 I l b 6 were obtained from Dr. H.J. Evans, Oregon State University, Corvallis, U.S.A.; USDA138 from Dr. H. Keyser, USDA, Beltsville, U.S.A.; 3.15B3 from the Facultd des Sciences Agronomics, Gembloux, Belgium; NU150 from Dr. J.M. Vincent, University of Sidney, Sidney, Australia; CB1003 from Dr. R.J. Roughley, Horticulture Research Station, Gosford, Australia. Cultures were maintained in yeast extract-mannitol slants [19] and stored at 4 ° C. Plant material and growth conditions Seeds of soybean (G. max L. cv. Amsoy), peas (P. sativum L. cv. Lincoln), blackeyed cowpeas (V. unguiculata L. Walp sup. unguiculata), green gram (V. radiata L. Wilczek), lentils (L. culinaris Medik.) and beans (V. faba L.) were surface disinfected [19] and germinated in 0.5% agar plates. After
193 3 days the germinated seeds were planted in autoclaved Leonard jars containing free-nitrogen plant nutrient solution [19]. Four seedlings per jar were inoculated with 10 ml of yeast extract-mannitol liquid culture of the appropriate Rhizobium strains. Plants were grown in a controlled environment chamber with a photoperiod of 15 h and irradiated with fluorescent and incandescent light at an intensity of 30 klux. The temperature during the day/night cycle was 27°/18°C for soybeans, cowpeas and green gram and 23°/15°C for peas, beans and lentils.
Assays of nodules Plants were harvested after 30 days of growth and nodules attached to segments of roots (2--5 cm) were excised. Acetylene reduction and H2 evolution rates were chromatographically determined in separate samples of nodules (0.2--0.4 g). Ethylene was measured as in Ref. 7 and H2 as described by Hanus et al. [ 20 ]. • Preparation and assay of bacteroids Bacteroids from each Rhizobium-legume combination were obtained from nodules collected from 5--10 Leonard jar culture units. Nodules were crushed and bacteroids prepared as described by Emerich et al. [11] except that anaerobic conditions were not maintained and final bacteroid pellet was suspended in 10 ml of Mg2+-phosphate buffer (50 mM potassium phosphate, 2.5 mM magnesium chloride, pH 7.0). Hydrogenase activity of the bacteroid suspensions was determined from the H~-uptake rates when H2 and an electron acceptor were externally supplied. Hydrogen and O~ in the aqueous phase were measured continuously and simultaneously by an amperometric technique [21]. Phenazine methosulfate (PMS) (2 mM) and methylene blue (1 mM) were used as artificial electron acceptors. Protein determinations The protein content of bacteroid suspensions were determined by digesting the cells according to Stickland [22] and using the micro-biuret method as described by Goa [23]. Bovin serum albumin was used as standard. Chemicals Hydrogen and O2 were purchased from Sociedad Espafiola de Oxygeno (Spain), C2H2 was generated from calcium carbide, PMS was obtained from Serva Feinbiochemica, F.R.G. and methylene blue from Merck AG, F.R.G. RESULTS In order to study the effect of the host on the expression of H2-oxidizing hydrogenases in legume nodules two characteristics are required in the Rhizobium strains to be used: (a) to possess the H2-uptake hydrogenase genes and (b) to be able to nodulate different host legumes.
194 Strain 128C53 of R. leguminosarum [9,10] and several strains of R. japonicurn [ 5, 7 ] have been reported to induce H2-uptake hydrogenases in peas and soybeans, respectively. In preliminary assays we tested the capacity of these strains to nodulate different legume species of the pea and cowpea rhizobial cross inoculation groups. Strain 128C53 was found to nodulate effectively lentils, beans and peas. All ten R. japonicum strains tested were able to effectively nodulate cowpeas, besides soybeans and six of them nodulated also green gram. Table I shows the relative efficiency of nitrogen fixation of nodules produced by strain 128C53 in peas, beans and lentils. Nodules from the three hosts evolved H2 in air. However when the rates of H2 evolution were compared with the C2H2 reduction rates as a measurement of the total nitrogenase activity, it was shown that while lentil nodules were loosing 49% o f the total energy available for nitrogenase, nodules of peas and beans were more efficient, losing only 17% and 36%, respectively. Bacteroids were extracted from the nodules and tested for hydrogenase activity with O2 and t w o non-physiological electron acceptors. The uptake of H2 externally supplied was followed amperometrically and Fig. 1 shows a typical recording obtained with bacteroids from pea nodules. Bacteroids from pea and bean nodules exhibited a capacity to consume H2 (Fig. 1 and Table I), indicating the presence of a H2-uptake system. However no detectable hydrogenase activity was found in bacteroids from lentil nodules, either with 02, PMS or methylene blue as final electron acceptors. Table II shows the relative efficiency of nodules and hydrogenase activities of bacteroids from eight H u p ÷ and t w o H u p - strains of R. japonicum in different hosts. With one exception, all Hup ÷ strains which evolved little or no H2 in soybean nodules, also evolved little or no H2 in the
TABLE I EFFICIENCY O F N I T R O G E N FIXATION O F N O D U L E S A N D H Y D R O G E N A S E ACTIVITY OF BACTEROIDS PRODUCED BY STRAIN 128C53 OF R. LEGUMINOSAR UM IN PEAS, BEANS AND LENTILS Legume host
Peas Beans Lentils
H~ produced in aira (~mol/h/g fresh nod. wt.)
4.4 7.5 23.2
C2H 2 reduced a (umol/h/g fresh nod. wt.)
26.3 20.5 47.9
Relative efficiency l-H2 aira
Hydrogenase activity of bacteroids (electron acceptors) b
C~H2 red.
02
PMS
MB
0.83 ± 0.04 0.64 ± 0.06 0.51 ± 0.07
0.81 0.53 <0.05
0.62 0.33 <0.05
0.73 0.32 <0.05
a Values are means of determinations made in 5 replicated plant cultures (±S.E.M.). Plants were harvested and measurements made after 30 days. b ~mol H 2 consumed/htmg protein. MB, methylene blue.
195 I
!
!
m c o
...
20
7 MB
'0
t-
0
',_ ..............
0®
"0
"r"
0
|
1 Time
I
2
I
3
4
(minutes'~
Fig. 1. Amperometric recordings of H z ( ) a n d O 2 ( - - - - - - ) consumption by bacteroids (1.30 mg protein) from pea nodules of R. le~mino~rum strMn 1 2 8 C 5 3 . A t the times indicated, H 2 ( 2 7 / ~ M ) , O: (13/~M) and methylene blue (1 raM) were added to the electrode chamber (2.5 ml) as Mg2+-phospl'mte buffer solutions.
other hosts tested. Relative efficiencies in these hosts ranged between 0.96 and 1.0. The bacteroids extracted from the nodules produced by all these strains in the different hosts exhibited hydrogenase activities of the same order. The exception was strain 3Ilb6, which evolved H2 in green gram at a rate 16-fold higher than in cowpeas and soybeans. Although hydrogenase activity was detected in green gram bacteroids from this strain, its rate was only 0.30/~mol/h/mg protein, versus 3.94 and 3.81/zmol/h/mg protein in soybean and cowpea, respectively. The low relative efficiencies of nodules and the low hydrogenase activities of bacteroids from green gram inoculated with strain 3Ilb6 were confirmed in three additional experiments. The two Hup- strains of R. japonicum 3.15B3 and USDA138 produced nodules in soybeans, cowpeas and green gram while evolved large amounts of H2 in air. Bacteroids from nodules of the three hosts showed no capacity to take up H2 externally supplied. Relative efficiencies however, markedly differ among hosts, cowpeas relative efficiencies being the lowest.
196
T A B L E II GROWTH AND ENERGY EFFICIENCY OF NITROGEN FIXATION OF SOYBEAN C O W P E A A N D G R E E N G ~ A M I N O C U L A T E D WITH H Y D R O G E N A S E - P O S I T I V E A N D H Y D R O G E N A S E - N E G A T I V E S T R A I N S O F R. JAPONICUM Strains
Hosts
H 2 evolution C 2 H 2 reduction a Relative in aira (umol/h/g efficiency b (umol/h/g fresh nod. wt.) fresh nod. wt.)
H2-uptake by bacteroids c
Hydrogenase positive USDA122DES
Soybeans Cowpeas Green gram
0.0 0.0 0.0
18.9 24.62 18.1
1.00 + 0.0 1.00 -+ 0.0 1.00 -+ 0.0
5.28 4.87 4.18
3Ilb110
Soybeans Cowpeas G r e e n gram
0.0 0.0 0.0
30.2 19.2 6.8
"1.00 ~ 0.0 1.00 ± 0.0 1.00 -+ 0.0
4.67 4.65 5.20
3Ilb6
Soybeans Cowpeas G r e e n gram
0.39 0.41 6.58
20.92 18.66 20.25
0.98 -+ 0 . 0 1 3.94 0.99 -+ 0 . 0 0 5 3.81 0.69 ± 0 . 0 1 0.30
NU150
Soybeans Cowpeas G r e e n gram
1.10 0.10 0.00
27.3 18.69 20.4
0 . 9 6 + 0.02 0.99 -+ 0.03 1.00 +- 0.0
3.15 5.93 4.45
CB1003
Soybeans Cowpeas
0.60 0.00
30.9 17.35
0.98 ± 0.01 1.00 -+ 0.0
3.08 5.78
3Ilb142
Soybeans Cowpeas
0.1 0.0
18.4 12.30
0.99 -+ 0 . 0 4 1.00 -+ 0.0
3.96 4.63
3Ilb143
Soybeans Cowpeas
0.0 0.0
16.30 16.86
1.00 -+ 0.0 1.00 ~ 0.0
3.88 5.44
USDA136
Soybeans Cowpeas
0.0 0.0
19.2 16.25
1.00 ± 0.0 1.00 ± 0.0
4.25 5.20
44.0 11.4 31.0
0.65 -+ 0 . 0 3 0.49 ± 0.06 0.75 -+ 0.05
0.0 0.0 0.0
20.7 11.12 24.5
0.77 ± 0 . 0 6 0 . 3 6 ± 0.08 0 . 6 4 ± 0.05
0.0 0.0 0.0
Hydrogenase negative 3.15B3
Soybeans Cowpeas G r e e n gram
15.2 5.6 8.66
USDA138
Soybeans Cowpeas G r e e n gram
4.28 6.82 8.89
a Values are m e a n s o f d e t e r m i n a t i o n s m a d e in 4 r e p l i c a t e d p l a n t cultures. P l a n t s were
h a r v e s t e d a n d m e a s u r e m e n t s m a d e a f t e r 33 d a y s o f g r o w t h . Values r e p o r t e d as 0.0 were < 0 . 0 5 ~,mol o f H 2 p r o d u c e d / h / g fresh n o d u l e weight. b I"H~ ( a i r ) / C 2 H ~ r e d u c e d . Values are m e a n s ± S . E . M . c ~ m o l o f H2 e o n s u m e d / h / m g p r o t e i n . Values r e p o r t e d as 0.0 were < 0 . 0 5 ~ m o l o f H2 consumed/h/rag protein.
197 DISCUSSION
Results in Table I clearly show a marked effect on the legume host on the relative efficiency and hydrogenase activity of nodules produced by R. leguminosarum strain 128C53. This strain, which was previously shown to be Hup ÷ in pea nodules [9,10], exhibited a H2-uptake capacity in bacteroids from pea and bean nodules, but not in bacteroids from lentil nodules. Since no H2-uptake was observed either with PMS or methylene blue as artificial electron acceptors, the lack of hydrogenase activity in bacteroids from lentils is likely due to the absence of an active hydrogenase rather than to a defect on the electron transport chain to O:. By estimating deuterium disappearance, Dixon [18] also found that strain ONA 311 of R. leguminosarum induced a H2-uptake hydrogenase in nodules of P. sativum but not in nodules of V. faba. A clear host effect on the relative efficiency and on the expression of hydrogenase of R. japonicum in nodules was not observed in seven out of eight Hup ÷ strains tested. The seven strains expressed similar levels of hydrogenase activity in bacteroids from nodules of soybeans and cowpeas, and three of them also in bacteroids from green gram (Table II). The nodules from all hosts evolved little or no H~ in air. Cm~er et al. [5] also found no differences in the energy efficiency of nodules produced in seven cultivars of soybeans by the Hup ÷ strain 3Ilb143 of R. japonicum. By contrast, strain 3Ilb6 induced 10-fold higher hydrogenase activity in bacteroids from soybean and cowpea nodules than in bacteroids from nodules of green gram, which produced large amounts of H2 in air. While this work was in progress two preliminary reports appeared which indicate a similar effect of the host: Gibson et al. [23] communicated a host controlled development of hydrogenase in nodules formed by Rhizobium sp strains CB756 and 32H1, and Keyser et al. [24] reported significantly higher rates of H~ evolution in soybean nodules than in cowpea nodules produced in both hosts by two strains of R. japonicum. These results show that the legume host effects the expression of the H~-uptake hydrogenase of Rhizobium in nodules, but this effect is closely dependent on the Rhizobium strain. The mechanism of this effect is unknown and perhaps it will remain so until we understand the exact function of the H2-uptake hydrogenases in the nitrogen fixing process. In vitro experiments with bacteroids show that the oxidation of externally supplied H~ by the H2-uptake system provides energy for nitrogenase [8,11 ]. Since hydrogenase induction in free-living cells of R. japonicum is regulated by the presence of carbon substrates in the culture medium [16,17], one may speculate that hydrogenase activity and expression in legume nodules are controlled by the quantity and quality of photosynthates supplied by the plant host to the nodules. As can be seen in Tables I and II, hydrogenase activity of bacteroids correlates with the relative efficiency of nodules. This fact provides
198 a d d i t i o n a l s u p p o r t f o r t h e p r o p o s e d role f o r t h e H 2 - u p t a k e h y d r o g e n a s e in l e g u m e n o d u l e s o f recycling t h e H2 g e n e r a t e d b y t h e N2-fixing s y s t e m [ 2 , 2 5 ] . E n e r g y efficiencies o f n i t r o g e n f i x a t i o n m a y be a f f e c t e d b y f a c t o r s o t h e r t h a n t h e p r e s e n c e o f H 2 - u p t a k e h y d r o g e n a s e s since t h e t w o H u p strains o f R. japonicum e x a m i n e d have significantly d i f f e r e n t relative efficiencies in s o y b e a n s , c o w p e a s and green gram. ACKNOWLEDGEMENTS We are grateful t o Drs. J. B u r t o n , H.J. Evans, H. K e y s e r , R.J. R o u g h l e y and J.M. V i n c e n t f o r p r o d d i n g Rhizobium strains. This r e s e a r c h was s u p p o r t e d b y a G r a n t f r o m F u n d a c i S n R a m S n Areces (Spain). REFERENCES
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199 25 H. Keyser, P. van Berkum and D. Weber, Nitrogen activity and hydrogen evolution by Rhizobium japonicum in symbiosis with siratro and cowpea, in: A.H. Gibson and W.E. Newton (Eds.), Proceedings of the Fourth International Symposium on Nitrogen Fixation, Australian Academy of Science, Canberra, 1981, p. 374. 26 H.J. Evans, D.W. Emerich, T. Ruiz-Argiieso, S.L. Albretch, R.J. Maier, F. Sympson and S.A. Russell, Hydrogen metabolism in legume nodules and rhizobia: some recent developments, in: H.G. Schlegel and K. Schneider (Eds.), Hydrogenases: Their Catalytic Activity Structure and Function, Erich Goltze, GSttingen, 1978, p. 287.