Oct 20, 1982 - SLOGER C, D BEZDICEK, R MILBERG, N BOONKERD 1975 Seasonal and diurnal variations in N2(C2H2)-fixing activity in field soybeans.
Plant Physiol. (1983) 71, 194-196 0032-0889/83/7 1/0194/03/$00.50/0
Short Communication
Cytochromes of Rhizobium japonicum 61A76 Bacteroids from Soybean Nodules1 Received for publication April 20, 1982 and in revised form October 20, 1982
DONALD L. KEISTER, SARAH S. MARSH, AND MEHRESHAN T. EL MOKADEM2 Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387 ABSTRACT Bacteroids of Rhizobium japonicum 61A76 were isolated from nodules of field-grown soybean plants by sucrose density gradient fractionation. The major cytochromes, aa3, b, c, and possibly o were present in the bacteroids throughout the active nitrogen-fixing life of the nodule. This is in contrast with previous reports using other R. japonicum strains in which cyotchromes aa3 and o were not found.
The major terminal hemeprotein oxidases of aerobically-grown cells of various species of rhizobia have been reported to be Cyt aa3 and o (2, 7, 12, 14, 17). Most of these reports also indicated that Cyt aa3 and o were not found in bacteroids of the corresponding species. Instead, new CO-binding hemeproteins including C552, P40, and P420 were found in bacteroids which were not present in cultured cells (1, 5, 12, 14). Preliminary experiments in our laboratory with bacteroids from nodules of greenhouse-grown plants inoculated with strain 61A65 indicated that Cyt aa3 and possibly o were present in bacteroids. To verify this observation, we inoculated field-grown plants with this strain and isolated bacteroids from nodules of various ages. Bacteroids from this R. japonicum strain retain Cyt aa3 and possibly o throughout the active nitrogen-fixing life of the nodule. MATERIALS AND METHODS R. japonicum 61A76 was grown in a yeast extract, gluconate, inorganic salts medium (10). Soybeans (Glycine max cv Williams) were field grown during the summer of 1981. The seeds were heavily inoculated with 61A76 by pouring a liquid culture diluted to contain about 108 cells/ml over the seeds in the rows. Uninoculated plots were interspersed with inoculated plots. At appropriate periods, the tap root nodules were harvested, washed, and frozen and kept at -70°C. The field plot had not been used previously for many years, if ever for soybeans. Thus, uninoculated control plants had an average of less than one tap root nodule per plant whereas inoculated plants had an average of 17, 23, and 29 tap root nodules at 29, 43, and 56 d, respectively. Bacteroids were prepared from nodules by grinding with mortar and pestle in the buffer described by Ching et al. (5). The ' Supported in part by the United States Department of Agriculture under agreement No. 59-2394-1-653-0. Contribution No. 785 from this
laboratory. 2 Present address: Woman's College, Ain Shams University, Heliopolis, Cairo, Egypt.
postnuclei supernatant from 15 g nodules was layered on 10 ml of 35% (w/v) sucrose in 50 mm K-phosphate, pH 7.5. After centrifuging for 25 min at 17,000g, the bacteroid pellet was resuspended with 20 ml of the original buffer. Six ml were layered on stepwise gradients prepared in 50 mm phosphate consisting of 2 ml 57% (w/w) sucrose, 16 ml 50%o sucrose, 12 ml 45% sucrose. The gradients were centrifuged at 22,000 rpm in a Beckman SW27 rotor for 4.25 h. The bacteroids collecting at the 45 to 50%o sucrose interface were harvested and washed with 50 mm phosphate buffer, pH 6.8. This fraction corresponds to the mature bacteroid fraction described by Ching et al. (5). The bacteroids were suspended in phosphate buffer containing 0.1 mM MgCl2 and 2 i,g/ ml DNase and ruptured by passage through the French pressure cell. The membrane fraction sedimenting at 183,000g (90 min) was isolated, washed, and used for analysis. Cyt content was determined by spectroscopy using the difference spectra between ferricyanide oxidized and dithionite reduced or was determined for the CO-reactive hemeproteins using the difference between dithionite + CO minus dithionite spectra. The extinction coefficients used were those used by Appleby (1, 2). L-Malate dehydrogenase was assayed by the method of Ochoa (15). Protein was determined by the Lowry method after solubilization with 0.5 N NaOH for 10 min at 0°C using BSA as the standard.
RESULTS AND DISCUSSION We have been using R. japonicum 61A76 for the isolation of mutants defective in terminal oxidase activity (8, 9). We observed that bacteroids of the wild-type strain isolated from greenhousegrown plants contained levels of Cyt aa3 similar to those found in cultured cells. This observation was not consistent with reports from other laboratories (2, 12, 14). Inasmuch as greenhouse conditions depart markedly from field conditions, we explored this observation further by inoculating field-grown plants heavily with strain 61A76 during the summer of 1981. The plot used had no recent history of soybean cultivation. Uninoculated plants had only a few nodules and most of them were on lateral roots. Therefore, we used only the earliest formed tap root nodules for these studies. Because an average of less than one nodule per plant was found in this root area of uninoculated plants, there is a high degree of certainty that the nodules contained bacteroids of strain 61A76. Ching et al. (5) reported that the bacteroid fraction isolated from 35-d-old soybean nodules could be separated into three fractions by density gradient centrifugation. They suggested that the heaviest fraction corresponded to 'bacteria,' the intermediate fraction corresponded to 'transforming bacteria,' and the lightest fraction corresponded to 'mature bacteroids.' The mature bacteroids were devoid of Cyt aa3 whereas the bacterial fraction and
transforming bacterial fraction contained this Cyt. Ching et al. used a commercial inoculant for their studies so that the rhizobial 194
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CYT OF R. JAPONICUM BACTEROIDS Table I. Cyt Content of the Soluble and Particulate Fraction of Bacteroids and Cultured Cells of R. japonicum 61A 76 Hemeprotein Content
Co-reactive
Particulate Nodule Age
Soluble
aa3
b
c
(c)
P450
Particulate d 29 43 56 Cultured cells
145 91 75 140
547 500 590 336
501 472 574 270
nmol hemeig protein 158 181 192 200
55 40 37 65
Soluble
46 41 68
18 44
34
66
bacterial fraction, and fraction IV bands at the 52 to 57% interface and is the bacterial fraction. Cultured 61A76 cells band at this interface. Quantitatively, this distribution differs from the results ofChing et al. who used 28 to 35-d-old nodules. Whereas, mature bacteroids represented 50%o or less of the total in their results, we found 92% in fraction II (Fig. 1). In the 43- and 56-d-old nodules, 99% were mature bacteroids. We used nodules which had been frozen for 5 la these studies. During the 1982 growing season, we repeated these studies using freshly harvested nodules. No significant difference c in the distribution was observed. a 4 For preparative work, we have used a slightly different gradient to facilitate separation and collection of the mature bacteroid fraction. We did not investigate fractions III or IV inasmuch as they were such a minor part of the total bacteroids. We did not survey nodules younger than 29 d. It is possible that the quantitative distribution may differ in younger nodules, but very little nitrogen is fixed by younger nodules (11, 16). The hemeprotein concentrations found in bacteroids prepared from various age nodules are shown in Table I. Cyt aa3 was present in bacteroids throughout their most active nitrogen-fixing period (4, 11, 13, 16), and the level of this Cyt in the 29-d bacteroids was approximately the same as was found in aerobically TUBE NUMBER grown cultured cells of 61A76. The level of this Cyt declined FIG. 1. Discontinuous sucrose density gradient separation of bacteroids somewhat as the nodules aged. Previous studies have suggested from soybean nodules inoculated with R. japonicum 61A76. Bacteroids that senescence of tap root nodules begins between 50 and 65 d. from nodules of 29-d-old plants were suspended in 5 ml buffer and were Later developing lateral root nodules probably have higher levels layered on a gradient consisting of 8 ml 45% (w/w) sucrose, 10 ml 50%o of Cyt aa3, but this has not been determined. Cyt b and c are found in somewhat higher levels than in cultured cells as has been sucrose, 8 ml 52% sucrose, and 5 ml 57% sucrose. All sucrose solutions were prepared in 50 mm K-phosphate, pH 7.5. The gradients were centrireported for other strains and species of rhizobial bacteroids (1, 2, fuged for 4.25 h at 90,000g in a Beckman SW27 rotor. This is a modifi- 12, 14). cation of the method of Ching et al. (5). The gradient was displaced with We have not included Cyt o in Table I. This Cyt is present in a dense sucrose solution and fractionated. One-ml fractions were collected. cultured cells and appears to be present in bacteroids in levels Specific gravity (D20) was measured with a refractometer. similar to those observed in cultured cells. However, the identification in bacteroids is not certain and what appears to be Cyt o in strain is unknown. In order to ascertain whether the Cyt aa3 which our CO-difference spectra may in fact be P420 (1). Further work is needed along these lines to identify Cyt o and P420. we found to be present in preliminary experiments could be Recently, it was reported that R leguminosarum bacteroids and ascribed to the bacterial and transforming bacterial fraction or to the bacteroids, we separated our bacteroid preparation into frac- Rhizobium sp. NGR231 from Parasponia nodules have Cyt aa3 tions corresponding to the densities described by Ching et al. A (3). We have also observed Cyt aa3 in bacteroids from nodules typical gradient of 29-d-old nodule bacteroids is shown in Figure inoculated with a commercial inoculum. Coupled with our results 1. Fraction I contains mitochondria and some bacteroids that are on R japonicum 61A76, it is apparent that Cyt aa3 may be more lighter than the major fraction (II). This fraction did not enter the widely distributed in bacteroids of various species and strains than 45% sucrose. Fraction II bands at the 45 to 50%o sucrose interface previously suspected. Experiments with additional strains are in and corresponds to the mature bacteroid fraction. Electron micro- progress. Preliminary results suggest that bacteroids of some scopic examination of this fraction demonstrated that it was strains have Cyt aa3 while others do not. Thus, it appears that substantially free of mitochondria. Enzymic assay of fraction II bacteroids may or may not contain Cyt aa3, and no direct correfor the marker enzyme for plant mitochondria, malate dehydro- lation can be ascribed to the development of the bacteroid state genase (6), showed that this fraction contained less than 2% of the and the presence or absence of this Cyt. To study the role of this total activity of the nodule homogenate. Fraction III bands at the and other Cyt in nodule respiration, we have isolated a series of 50 to 52% sucrose interface and corresponds to the transforming Cyt c- and aa3-deficient mutants. Preliminary studies on the 00
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KEISTER ET AL.
aerobic physiology of these mutants have been reported (8, 9). Acknowledgment-We thank W. R. Evans and D. K. Crist for help and advice during these experiments, and Harry Calvert and Mark Pence for electron microscopic examination of our samples. LITERATURE CITED 1. APPLEBY CA 1969 Electron transport systems of Rhizobium japonicum. I. Haemoprotein P4es, other CO-reactive pigments, cytochromes and oxidases in bacteroids from N2fixing root nodules. Biochim Biophys Acta 172: 71-87 2. APPLEBY CA 1969 Electron transport systems of Rhizobium japonicm. II. Rhizobnwm haemoglobin, cytochromes and oxidases in free-living (cultured) cells. Biochim Biophys Acta 172: 88-105 3. APPLEBY CA, FJ BERGERSEN, TM CHING, AH GIBSON, PM GRESSHOFF, MJ TRINICK 1981 Cytochromes of rhizobia from parasponia, pea and soybean nodules, and from nitrogen fixing continuous cultures. In AH Gibson, WE Newton, eds, Current Perspectives in Nitrogen Fixation. Australian Academy of Science, Canberra, p 369 4. BERGERSEN, FJ 1958 The bacterial component of soybean root nodules. J Gen Microbiol 19: 312-323 5. CHING TM, S HEDTKE, W NEWCOMB 1977 Isolation of bacteria, transforming bacteria, and bacteroids from soybean nodules. Plant Physiol 60: 771-774 6. CUTTING JA, HM SCHULMAN 1969 The site of heme synthesis in soybean root nodules. Biochim Biophys Acta 192: 486-493 7. DE HOLLANDER JA, AH STOUTHAMER 1980 The electron transport chain of Rhizobium trifolii. Eur J Biochem 111: 473-478
Plant Physiol. Vol. 71, 1983
8. EL MOKADEM MT, DL KEISTER 1982 Electron transport in Rhizobiumjaponicum. Isolation of cytochrome c deficient mutants. Israel J Bot 30: In press 9. EL MOKADEM MT, DL KEISTER 1982 Mutants of Rhizobiumjaponicum deficient in cytochromes c and aa3. Studies with aerobically grown cells. In JHG Stephens, ed, Proceedings of the 8th North American Rhizobium Conference. In press 10. FUCHS RL, DL KEISTER 1980 Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp. J Bacteriol 144: 641-648 11. HARDY RWF, RD HOLSTEN, EK JACKSON, RC BURNS 1968 The acetyleneethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 43: 1185-1207 12. KRETovICH WL, VI ROMANOV, AV KOROLYOV 1973 Rhizobium leguminosarum cytochromes ( Viciafaba). Plant Soil 39: 619-634 13. KLUCAS RV, D ARP 1977 Physiological and biochemical studies on senescing tap root nodules of soybeans. Can J Microbiol 23: 1426-1432 14. MATUs VK, SS MELIK-SARKISYAN, VL KRETovICH 1973 Cytochromes and respiration rate of bacteroids from nodules of lupine inoculated with effective and ineffective strains of Rhizobium lupini. Microbiology (Engl Transl Mikrobiologiya) 42: 95-100 15. OCHOA S 1955 Malic dehydrogenase from pig heart. Methods Enzymol 1: 735736 16. SLOGER C, D BEZDICEK, R MILBERG, N BOONKERD 1975 Seasonal and diurnal variations in N2(C2H2)-fixing activity in field soybeans. In WDP Steward, ed, Nitrogen Fixation by Free-Living Microorganisms. Cambridge University Press, Cambridge, pp 271-184 17. TuzImuRA K, I WATANABE 1964 Electron transport systems of Rhizobium grown in nodules and in laboratory medium. Plant Cell Physiol 5: 157-170
