Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules

Planta (1981)152:534-543 P l ~ H t ~ �9 Springer-Verlag 1981

Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules

F.J. Bergersen and C.A. Appleby CSIRO, Division of Plant Industry, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia

Abstract. Methods are reported for the preparation from soybean (Glycine max (L.) Merr.) root nodules, of well-washed, intact membrane envelopes containing bacteroids. The intact envelopes are of much lower density than the bacteroids within and therefore only low speed centrifugation (approx. 150 g) may be used. The optimum osmotic strength is 600 mOsm/ kg H20. The envelope contents were recovered following mild osmotic shock and-or hard centrifugal packing at > 10,000 g. Extracts prepared in this way contained leghaemoglobin (identified spectrophotometrically), low-molecular-weight fluorescent materials and other components which are yet to be identified. Envelope leghaemoglobin did not react with specific antibody until the envelopes were ruptured. 131I-Labelled leghaemoglobin or bovine serum albumin, added during initial breakage of nodule cells, was not released when envelopes were ruptured to release leghaemoglobin. It is therefore concluded that this leghaemoglobin is located within the envelope space and did not arise from adhering or occluded cytosol leghaemoglobin. Based on the number and dimensions of microscopically intact envelopes in these preparations, the concentration within that space was in the range 178-523 pM. Based on these estimates, leghaemoglobin within envelopes represented about one third of the total amount present in the nodule cells. Flat-bed isoelectric focusing of partially-purified envelope leghaemoglobin demonstrated that the latter contained all of the leghaemoglobin components previously reported for soybean nodules and an additional minor component focusing between leghaemoglobins a and b.

Key words: Bacteroids Glycine Leghaemoglobin - Membrane envelopes (root nodules) - Root nodules.

Introduction

The nitrogen-fixing bacteroids of soybean root nodules are located, in groups, within membrane- bounded vesicles or envelopes (Bergersen and Briggs 1958; Goodchild and Bergersen 1966; Bergersen and Goodchild 1973; Werner and M6rschel 1978). The membrane envelope (sometimes referred to as the peri-bacteroid membrane, e.g. Robertson et al. 1978) arises from the host cell membrane following endocy- totic processes (Dixon 1967; Goodchild and Berger- sen 1966; Tu 1974; Bassett etal. 1977). There are about 10,000 envelopes in each mature soybean nodule cell and the total space within the envelopes, but outside the bacteroids amounts to 46% of the volume of nodule cells aged 30 d (Bergersen and Goodchild 1973). The envelope space is therefore an important feature of nodule structure when the supply of nu- trients to and removal of products from the bacteroids are being considered.

Smith (1949) established that leghaemoglobin was confined to the bacteroid-containing cells of the cen- tral tissue of soybean nodules, but its intracellular location has been controversial (see review by Berger- sen 1980). Dilworth and Kidby (1968) used 59Fe and autoradiographic electron microscopy and concluded that most Fe was located between the membrane envelopes and the bacteroids, in nodules of serradella. There was insufficient Fe in any other compartment to account for the leghaemoglobin Fe, known by analysis to be present in the tissue. In soybean nodules, staining of haemoproteins in unfixed nodule slices by pre-oxidized diaminobenzidine, followed by opti- cal microscopy, demonstrated the presence of dis- crete, stained bodies whose size and distribution could only have been those of the membrane envelopes and their contents (Bergersen and Goodchild 1973). Tru- chet (1972), using electron-microscopic cytochem-

0032-0935/81/0152/0534/$02.00

F.J. Bergersen and C.A. Appleby: Leghaemoglobin in bacteroid envelopes of soybean 535

istry, found that peroxidase activity of the leghaemoglobin of pea nodules was primarily located between the bacteroid surfaces and the enclosing membrane envelopes. However, the activity was unevenly distrib- uted in this location. Gourret and Fernandez-Arias (1974) studied nodules of Trifolium repens with similar methods, but stained their tissue at pH 9 and found that the space between bacteroids and enclosing membrane envelope was intensely stained. They concluded, from comparisons with suitable controls, that this was the result of pseudoperoxidase activity of denatured (fixed) leghaemoglobin. However, other electron-microscope studies have yielded contrary evidence, that is, the leghaemoglobin seemed to be confined to the host cytoplasm between the membrane envelopes. Dart and Chandler (1971) used X-ray mi- croprobe techniques for Fe, and Verma and Bal (1976) and Verma et al. (1978) used ferritin-labelled antiserum prepared against purified leghaemoglobin. Some of these results were critically reviewed by Goodchild (1977).

Several authors have reported the preparation of membrane envelopes containing bacteroids by centrifugal fractionation of nodule homogenates on sucrose density gradients (Robertson et al. 1978, lupin nodules; Verma et al. 1978, soybean root nodules). These preparations contained no detectable leghaemoglobin. In addition Robertson et al. (1978) produced evidence that the envelopes were still intact, since ferritin added to the preparations had not penetrated inside the envelopes when they were subsequently examined by electron microscopy. The consistent failure to find leghaemoglobin within membrane envelopes in such preparations, coupled with the fact that this protein is synthesized in the host cytoplasm, is considered by these authors to be conclusive evidence that leghaemoglobin is confined to the host cytoplasm. However, a recent report (Livanova et al. 1979) describes the preparation of bacteroid- and leghaemoglobin-containing membrane envelopes from lupin nodules.

It is important for a more complete understanding of the role of leghaemoglobin in nodules that its intracellular location(s) be established (Bergersen 1980). We now report the preparation from soybean root nodule cells, of well-washed intact bacteroid-containing membrane envelopes from which the contents can be recovered for study. Leghaemoglobin is shown to be a prominent component of the envelope contents.

Material and methods

Nodules. Soybeans (Glycine max (L.) Merr. cv. Lincoln, fiom seed produced in this laboratory) inoculated with Rhizobium japonicum strain CB1809 were grown as described by Bergersen and Tinher (1970). Nodules (28-30 d after they were first visible on the roots) were picked from washed roots, washed again, and blotted dry.

Preparation of envelopes. Washed nodules (20-25 g fresh weight) were sliced with a sharp scalpel blade into breakage medium (25 ml) with the following composition: mannitol, 4.55% (w/v); sorbitol, 2.28%; dextran T40 (Pharmacia Fine Chemicals, Uppsala, Sweden), 4%; polyvinylpyrrolidone (Kollidon 25; BASF, Austra- lia, Sydney), 1%; bovine serum albumin, 0.5%; CaC12, 0.015%; MgCla-7H20, 0.02%; all dissolved in 150mM imidazole-HC1 buffer, pH 7.4; 600 mOsm/kg HzO (osmolarity measured by Halb- mikroosmometer, Knauer & Co., Berlin, Germany). The sliced nodules were crushed gently in a glass mortar and pestle and the macerate filtered through MiracIoth (Chicopee Mills, Milltown, N.J., USA), which had been previously moistened with the breakage medium, into glass centrifuge tubes. The crushed nodules were irrigated on the filter with breakage medium until the filtrate to- talled 40 ml. The combined filtrates were then centrifi~ged in a swinging bucket rotor for 20 30 rain at 100-160 g at room tempera- ture (22 ~ C). The pellet consisted of a lower, pale-pink layer of naked bacteroids, overlaid by a very loosely packed, orange-brown layer of intact envelopes. The red-brown, turbid supernatant ($1) contained soluble leghaemoglobin, small particles and mitochon- dria, and was discarded or used for other purposes. The pellet was then washed once with 10 volumes of breakage medium, using the same conditions in the centrifuge, and then washed twice (10 volumes each) with an isotonic washing solution of lower viscosity (10% w/v mannitol, 0.015% CaCI2, 0.02% MgC12.7H20, dissolved in 15 raM imidazole-HC1 buffer pH 7.4; 600 mOsm/kg HzO). In this solution the intact envelopes were sedimented in 5-7 rain at 120 g, leaving most of the bacteroids in the supernatant.

Extraction of envelopes. The loosely packed pellets of envelopes were resuspended in 2.5 volumes of washing solution and ruptured by centrifugation at 10,000 20,000 g for 10 min. The pellet was then dispersed in the supernatant and the centrifuging repeated. The supernatant was orange-brown. Alternatively the washed envelopes were suspended in 2.5 volumes of 15 mM imidazole buffer or 50 mM K2HPO4-KHzPO 4 buffer, pH 7.4 and then centrifuged at 10,000-20,000 g. Generally, extraction was superior using the second procedure, presumably because the osmotic shock was more effective than centrifuging alone for rupturing the envelopes.

Percollgradients. Percoll (Pharmacia), 10 ml, was mixed with 10 ml of twice-concentrated washing solution, adjusted to pH 7.4 with HCI, and the mixture divided between two capped Spinco tubes, 7.5 cm long, 1.5 cm diameter. The gradients were formed by centrifugation at 20,000 rpm for 20 rain using a Model L centrifuge and the TY65 rotor (Spinco Division, Beckman Instruments, Palo Alto, Ca1., USA). The pellet (free bacteroids plus intact envelopes) from the first wash in breakage medium was resuspended in 2.5 volumes of washing solution and 1 ml layered on the top of one tube of the Percoll gradient. One ml of washing solution was layered in the other tube. Both tubes were centrifuged at 1,000 rpm for 30 rain. The blank tube was used to calibrate the gradients using coloured density-marker beads (Pharmacia).

Microscopy. At all stages during preparation, the integrity of the envelopes was checked by bright-field and phase-contrast microscopy. Counts of envelope numbers were made using dilutions in isotonic media in a Petroff-Hauser bacterial counting chamber (Hauser, Philadelphia, Pa., USA). Envelope dimensions were measured directly nsing a calibrated ocular micrometer and from electron micrographs.

For transmission electron microscopy washed envelopes were fixed for 1 h at 23 ~ C in an isotonic medium containing K2HPOa- KH2PO4 buffer (50 raM, pH 7.0), 3% glutaraIdehyde, 4.55% mannitol, 2.275% sorbitol, followed by embedding and sectioning pro- cedures described previously (Bergersen and Goodchild 1973).

Methods using antiserum. Antiserum was prepared in rabbits as described by Schwinghamer and Dudman (1980) using highly purl-

536 F.J. Bergersen and C.A. Appleby: Leghaemoglobin in bacteroid envelopes of soybean

fied leghaemoglobin c (Appleby 1974) as antigen. This antigen is now known to contain three leghaemoglobins, viz. Lbcl, Lbc2, Lbe3 (Fuchsman and Appleby 1979). The antiserum reacts with all leghaemoglobin components from soybean nodules (W.F. Dud- man, CSIRO, Canberra, personal communication) and the maximum precipitate with 0.2 ml was formed with 5 gg of purified mixed leghaemoglobin prepared by the method of Appleby and Bergersen (1980). Up to this equivalence point, there was a linear relationship between weight of precipitate formed and weight of antigen added. Amounts of reactant were adjusted to be within this range. The procedure was to add antiserum to a preparation, incubate for 1 h at 25 ~ C with gentle shaking, clarify by centrifugation and then to estimate the antibody remaining in the supernatant by back titration with the leghaemoglobin antigen. Envelope resi- dues from which all leghaemoglobin had been removed, were included to correct for small nonspecific binding of antibody. Amounts of antigen originally present were then determined from a standard curve constructed from data for assays containing known amounts of leghaemoglobin.

Experiments with 1311-labelled proteins. Bovine serum albumin (fraction V, 0.5 mg/ml; Sigma Chemical Co., St. Louis, Mo., USA) and purified soybean leghaemoglobin (Appleby and Bergersen 1980) were intensely labelled with 131I (New England Nuclear, Boston, Mass., USA) as described by McConahey and Dixon (1980). Labelled protein (0.1 ml containing about 55.5.104 Bq> 15 ~tCi of 131I) was added to breakage medium immediately before crushing the nodule slices. Removal of radioactivity during washing and breakage of membrane envelopes was monitored by precipitat- ing the protein in fractions with 10% trichloroaeetic acid (0.2 ml ml 1 of fraction), centrifuging at l l ,000g for 10rain, draining the peliets, and then homogenizing them in 10 ml of a scintillation mixture 2,5-diphenyloxazole, 6 g 1,4-bis [2-(5-phenyloxazolyl)]-ben- zene, 0.6 g; toluene, 670 ml; Triton X-100, 330 ml and counting disintegrations in a liquid scintillation spectrometer.

Spectrophotometry. Routine measurements of leghaemoglobin in envelope extracts were made from reduced (Na2S204) + CO minus reduced difference spectra recorded with a Model 635 spectrophotometer (Varian-Techtron, Melbourne, Australia), using standards of purified mixed leghaemoglobin (Appleby and Bergersen 1980). Spectra of partially purified envelope-extract components were recorded with a Model 557 spectrophotometer (Hitachi-Perkin Elmer, Tokyo, Japan). Fluorescence spectra (excitation and emission) were recorded with a model MPF-44B spectrofluorimeter (Perkin Elmer, Norwalk, Conn., USA).

Fractionation of the components of envelope extract. Envelope extracts were concentrated by pressure filtration over PM10 membranes (Amicon, Lexington, Mass., USA). The buffer was ex- changed three times by adding column buffer and reconcentrating. The concentrated extract (0.5 ml) was fractionated on a column (14.5 cm long, 1 cm diameter) of Sephadex G75 (superfine; Phar- macia), equilibrated with 50raM K2HPO4-KHzPO~, pH7.4, containing 10 mM nicotinic acid. Fractions of 0.6 ml were collected and spectra recorded using column buffer as reference. The fractions containing leghaemoglobin were combined and concentrated for isoelectric focusing.

Flat-bed isoelectric focusing. Leghaemogiobin was resolved into the component proteins as the nicotinate complexes, on LKB 1804- 111, pH 4-5, analytical polyacrylamide gels (LKB-Produkter, Bromma, Sweden), as described by Fuchsman and Appleby (1979). Focused gels were cut into individual tracks, mounted in buffer in a quartz boat, and scanned in a Varian Techtron 635 spectrophotometer~ fitted with gel-scanning accessory and masks as described previously (Fuchsman and Appleby 1979). The scanning wave-

length was the Soret absorbance peak for leghaemoglobin nicotinate (407 nm) or for strong bands, the Sorer peak shoulder (422- 435 nm). Spectra of individual bands were recorded in situ at the position of maximum scan absorbance, or following excision and mounting of the bands in a suitably masked, demountable cuvette ; blank gel was used as reference.

Results and discussion

Properties of envelope preparations. The microscopic appearance of the envelope preparations is illustrated in Fig. 1. The isolated envelopes were similar in structure and appearance to those seen in transmission electron micrographs of soybean nodule tissue. The pellet volume of bacteroids, released from washed envelope preparations at extraction and centrifugally packed, indicated that up to about one third of the bacteroids originally present were finally recovered in the best preparations of washed intact envelopes. The results of Percoll gradient fractionations (Fig. 2) show that the density of intact envelopes is much lower than that of free bacteroids. The values given are probably not accurate, as they are based on cali- bration with density-marker beads, whose density refers to use with sucrose rather than the mixed osmo- ticum used in these experiments. For example, the free bacteroids appeared to have a density of about 1.145 g cm -3, whereas in other work (Ching and Hedtke 1977)values of 1.22-1.27gcm -3 were obtained for" soybean bacteroids. However, the quite large difference in apparent density between intact envelopes and the bacteroids contained within them, indicates that a substantial amount of lower-density material is also present within envelopes, and also explains why it was necessary to use very low centrifuge speeds to obtain yields of intact envelopes which were sufficiently large for further analysis. Percoll gradients were not suitable for preparative purposes of the bulky nature of the envelope fraction and the relatively low yields of intact envelopes.

Identification of leghaemoglobin in envelope extracts. Figure 3 a illustrates the absorption spectra of an envelope extract prepared in 15 mM imidazole-HC1 buffer, pH 7.4, and the spectrum of reduced, purified total nodule leghaemoglobin in the same buffer is shown for comparison. Although envelope extracts clearly contain other materials (e.g. the broad shoulder near 470 nm in curve A and the broadened peak near 550 nm in curve B), the presence of leghaemoglobin is evident. This is confirmed by the more specific reduced + CO minus reduced difference spectra in Fig. 3b; this method was used throughout to quantify leghaemoglobin in envelope extracts. Enve- lope extracts, prepared in the proportions described in Material and methods, invariably contained 2-


Fig. 1. Microscopical appearance of washed envelope preparations from soybean nodules. Transmission electron micrograph of a fixed, embedded and sectioned envelope preparation. Bar= 1 gin, x i3,000

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3.5 pM leghaemoglobin (estimated by CO difference spectra) before concentration.

Removal of leghaemoglobin during washing of envelopes. Figure 4 illustrates the course of removal of leghaemoglobin from an envelope preparat ion during washing and following breakage. The rate of removal is slightly greater than expected f rom the calculated amounts of supernatant occluded in the pellet. This finding indicates that there may be continual damage during washing or that adsorbed leghaemoglobin is being removed in addition to that occluded in the pellet. Following osmotic shock and hard centrifugal packing, the concentration of leghaemoglobin was about 100 times greater in the supernatant than expected f rom the residue measured in the previous wash. Thereafter leghaemoglobin was removed at the same rate as before breakage. These results indicate that leghaemoglobin was present free in the envelope space and was released upon breakage of the membrane by osmotic shock and centrifugal packing. This conclusion is supported by the results of experiments with antibody specific for leghaemoglobin.


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Antibody experiment. Table 1 summarizes the results of experiments which showed that in washed, intact envelopes there was little leghaemoglobin reacting with added antiserum. When the envelopes were ruptured by osmotic shock, the amount of antibody removed was equivalent to the amount of leghaemoglobin shown by spectrophotometry to be released. These results constitute strong evidence that the leghaemoglobin released from washed preparations of envelopes is within the envelopes and not adhering to the outer surfaces, where it would be expected to react with added antibody.

SUPERNATANT No.

Fig. 4. Removal of leghaemoglobin at breakage of soybean nodules, during washing of envelopes and following breakage of envelopes. For details, see Material and methods

Table 1. Estimation of immune-reactive leghaemoglobin in envelope preparations from soybean root nodules. A washed envelope preparation was suspended in isotonic medium and divided into 1 ml portions. The following duplicated treatments were applied:

A. Control, no further treatment. B. The suspension was centrifuged at 100 g and the envelopes

broken by osmotic shock and centrifugation at 15,000g, as in Material and Methods. The pellet was then resuspended in the supernatant.

C. As in B, but the pellet was washed three times to remove released leghaemoglobin and resuspended in buffered saline. It contained membrane fragments and bacteroids.

Immune-reactive leghaemoglobin in each treatment was then estimated as described in Material and methods, following addition of specific antiserum. The extracted leghaemoglobin was estimated at 8.1 lag/ml or original suspension

Treatment Residual antibody Calculated (lag immune- leghaemoglobin precipitate in initial protein) reaction (gg)

A. Intact envelopes 76.3 1.7 70.8

B. Broken envelopes 6.6 8.7 a 8.0

a Corrected for antibody bound non-specifically by broken envelopes and bacteroids in treatment C

Livanova et al. (1979) reported similar results with lupin nodules, using immune-diffusion reactions in agar, and concluded that leghaemoglobin was present inside the bacteroid-enclosing membrane.


Experiments with 131I-labelled proteins added during envelope preparation. It might be argued that leghaemoglobin recovered from within the envelope space resulted from the envelopes opening during tissue breakage or during centrifugation, then resealing, thus enclosing leghaemoglobin and other cytoplasmic components. Microscopical observation of envelope preparations showed that envelope breakage caused by mechanical pressure on the cover glass, or during drying out, resulted in the extrusion of bacteroids and the formation of membrane vesicles which were much smaller than intact envelopes and did not contain bacteroids. Vesicles of this type remained in suspension at the low centrifugal speeds used to sediment bacteroid-containing envelopes. Further evidence against a possible cytosol origin of envelope leghaemoglobin was obtained with the use of 131I-labelled proteins, added to breakage medium just prior to breakage of the nodule cells. The exogenous protein was very highly labelled and large numbers of radioactive counts were obtained after eight cycles of centrifugal washing. Preliminary results established that a little labelled bovine serum albumin adhered to the

membrane residue (about 0.6-0.8% of the total radioactivity supplied). Bovine serum albumin had been included in the breakage medium because it increased the stability of the membrane envelopes. This effect seems to be associated with binding of the protein by the membrane, since it was not removed by pro- longed washing after breakage of the envelopes, although changed ionic strength between the washing medium (containing 15mM imidazole-HC1) and 50 mM phosphate at envelope breakage, removed a small proportion of this bound radioactive protein. This desorption effect was eliminated when the envelope preparation was washed with breakage medium at all steps prior to rupturing the envelopes. Data for a typical experiment are shown in Fig. 5. Leghae- moglobin (50 pg) labelled with 131I was added just before the nodule tissue was broken. During washing (Fig. 5a) radioactivity and leghaemoglobin were removed at similar rates. The percentage values of Fig. 5a relate to quantities present in the filtrate of broken nodules. In Fig. 5b, data are presented for the extraction of leghaemoglobin and radioactivity from a sample of washed envelopes. The percentage

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Fig. 5a, b. Removal of leghaemoglobin and t31I-labelled leghaemoglobin (a) during washing and (b) following breakage of bacteroid- containing envelopes from soybean root nodules. Nodules (10.2 g fresh wt) were sliced into 10 ml of breakage medium and 0.1 ml of leghaemoglobin (0.5 mg/ml) labelled with t3~I (approx. 55.5.104 Bq) was added before crushing. Preparation and washing of envelopes was as described in Material and methods but washing was with 10 ml volumes of breakage medium. (a) Radioactivity (�9 and leghaemoglobin (e) were measured at each step during washing and values were plotted as a percentage of the totals initially present ,(4.243 lamol of leghaemoglobin and 15,081,016 cpm). (b) A sample of the suspension of washed envelopes was centrifuged, broken by resuspension in 3.5 ml of 50 mM K-phosphate, pH 7.4, and centrifugation at 11,000 g. The residue was resuspended and washed twice with 3.5 ml volumes of the same buffer. Each extract was centrifuged at 100,000 g for 10 min before radioactive protein and leghaemoglobin were measured for each step. Values are plotted as percentages of the totals present in the washed envelope sample (17.57 nmol of leghaemoglobin and 107,346 cpm)


values relate to quantities present before the final wash. Following rupture of the envelopes, 71% of the leghaemoglobin present appeared in the supernatant after centrifugation. For radioactivity the value was 2.6%. Further, the rate of removal of radioactivity was not perturbed by envelope rupture and the rate at which radioactivity had been removed during envelope washing continued when ruptured envelopes were washed (Fig. 5). The removal of leghaemoglobin followed a very different course after rupture of the envelopes. The data show clearly that no additional radioactivity was released when rupture of the envelopes released the leghaemoglobin contained within them. Essentially identical results were obtained when 13 l i_labelled bovine serum albumin replaced labelled leghaemoglobin added prior to breakage of nodule tissue. Thus, the envelope leghaemoglobin could not have arisen from occlusion of cytosol leghaemoglobin during preparation. In this experiment (Fig. 5) the envelope residue accounted for 85% of the radioactivity of washed envelopes, This residue corltained no spectrophotometrically detectable leghaemoglobin, but adhering protein may account for the small amount of antileghaemoglobin-reactive material remaining in washed envelopes from which all leghaemoglobin appeared to have been removed (Table 1, treatment C).

The concentration of leghaemoglobin in washed envelopes. The concentration of leghaemoglobin within envelopes was calculated as follows. The number of envelopes present in a preparation was estimated from microscopic counts of dilutions of samples. Then the volume of intramembrane space was calculated from the average dimensions and bacteroid numbers and volume per envelope measured microscopically and from electron micrographs of envelope preparations. The moles of leghaemoglobin extracted, divided by the calculated volume of the intra-membrane space, gave an estimate of concentration (Table 2). The average value was 342 gM, but the range of values was quite large.

Leghaemoglobins in envelope extracts. In soybean root nodules, eight different leghaemoglobin components have been recognized by flat-bed isoelectric focusing of the nicotinate complexes (Fuchsman and Appleby 1979). Envelope-extract leghaemoglobin, partially purified by repeated pressure filtration and Sephadex G75 chromatography, was concentrated and sepa- rated into components by the same method. For comparison, companion tracks of highly purified total nodule leghaemoglobin and of $1 (see Material and methods) leghaemoglobin were run. Results of typical scans are shown in Fig. 6. There was no difference

Table 2. Calculation of leghaemoglobin concentration within washed envelopes from soybean root nodules, based on numbers of intact envelopes and their dimensions, the number and dimensions of bacteroids and amounts of leghaemoglobin present in washed envelope preparations.

Data for three different experiments

Leghaemo- Total No. Total Leghaemo- globin intact envelope globin extracted enve lopes concentration (gM) (nmol) space (cm3) a

6.10 1.87.109 18.7.10 -3 326 9.06 1.73.109 17.3.10-3 523 2.72 1.53.109 15.3" 10 -3 178

2=342

a From electron microscopy ofenvelope preparations. The average envelope diameter was 3.41-10-4 cm with an average of 8.7 bacteroids per envelope. Direct microscope measurements gave a mean envelope diameter of 3.8.10 -4 cm (SD =0.75.10 -~ cm). The dimensions of bacteroids by electron microscopy of shad- owed preparations gave a mean volume of 1.22.10-12 cm 3 per bacteroid (SD =0.40.10-12 cm3). From these values the average envelope space was calculated to be 10.0.10 12 cm 3. (This com- pares with the values of 9.1 �9 10- ~2 cm 3 for the data for younger nodules given in Bergersen and Goodchild 1973)

between Sj and purified total leghaemoglobins. Enve- lope-extract leghaemoglobin also contained all of the components in similar proportions to those previously reported for soybean nodules of the same age (Fuchs- man and Appleby 1979; Fig. 6, Table 3) but in addition there was a minor band focusing between leghaemoglobins a and b (Fig. 6 b). This band had a typical leghaemoglobin nicotinate spectrum, as did all leghaemoglobin bands. Envelope-extract tracks also had a br ight red band which moved to the cathode. When purified from envelope extracts by preparative isoelectric focusing (using pH 9-11 Ampholine), this cathode band was found to contain a cytochrome c-like protein, whose isoelectric point was near pH 9.7. The Soret absorption characteristics indicated that this protein was different from both soybean-bacteroid cytochrome c and plant-mitochondrial cytochrome c. The cathode band was not detected in purified total leghaemoglobin, or when $1 preparations were examined by the same isoelectric focusing techniques in parallel tracks.

Other envelope-extract components. Pressure filtrates (passing PM10 membranes) of envelope extracts were yellow and contained three fluorescent materials. Ex- citation and emission spectra are shown in Fig. 7. The excitation and fluorescence peaks at 452 and 530 nm respectively, indicate the presence of riboflavin, which has been reported previously in nodules (Pankhurst et al. 1974). The two other fluorescent materials have not yet been identified. Envelope ex-


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Fig. 6a, b. Leghaemoglobin components in (a) partially purified envelope extract and in (b) purified total leghaemoglobin from soybean root nodules. Scanning traces of isoelectric focusing in flat-bed gels. Minor components on scale when scanned at 407 nm (Soret peak; dashed lines) and major components when scanned at 422 and 435 nm respectively (continuous lines). The vertical bar indicates the absorbance scale

Table 3. Proportions of leghaemoglobins in extracts of bacteroid-containing envelopes prepared from soybean root nodules compared with purified total nodule leghaemoglobin. Areas of peaks of scans of isoelectric focusing gels were estimated by weighing. Minor components are expressed as a ratio of the corresponding major component. Major components are expressed as a ratio of leghaemoglobin ca. Values in ( ) are standard deviations of the mean of three or four separate experiments. The unique envelope band marked with an arrow in Fig. 5b is labelled x

Preparation Ratio

Major components Minor components

ale3 c,/ca c=/c~ b/a x/a dt/cl d2/cz d3/c3

Envelope leghaemoglobin 2.32 0.85 (0.06) (0.05)

Total purified leghaemoglobin 2.17 0.91 (0.17) (0.18)

0.66 0.053 0.027 0.12 0.14 0.14 (0.07) (0.004) (0.003) (0.01) (0.01) (0.01)

0.65 0.055 - 0.12 0.16 0.17 (0.05) (0.003) -- (0.02) (0.01) (0.01)

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5o

~" 40

3O

20

10

b 365

,' \ / \ tt ~ J

/ \, r l \

, \ 290 344 [ \

,k// '- / \ i \ , ' ' I ' / ' : \ . . . . " ....

oL 1.

200 240 280 320 360 400 440 480 520 560 600 640 680

.& (nm)

Fig. 7a, b. Excitation and emission spectra of low-molecular-weight materials removed from envelope extracts by pressure filtration over an Amicon PM10 membrane, a Excitation spectrum for fluorescence at 600 nm ( - - ) and fluorescence spectrum for excitation at 450nm ( . . . . . ). b Excitation spectra for fluorescence at 452nm (- ) and 340rim ( . . . . ); fluorescence spectra for excitation at 350 n m ( . . . . . ) and 290 n m ( . . . . . )


tracts also contain other higher-molecular-weight materials, seen in fractions from G75 columns. They will be described in a later communication.

General discussion. The preparation of intact envelopes was conditional upon the use of low centrifugal speeds (because of the density difference between the intact envelopes and the bacteroids within), the use of media with low concentrations of monovalent cat- ions (cf. Livanova et al. 1979), and careful matching of osmolarity of all solutions used. Initial damage when breaking the nodule tissue is a major limitation on yield of envelopes; even blunt versus new scalpel blades for slicing make a perceptible difference.

The results showed that only a small proportion of the nodule leghaemoglobin was recovered in washed envelopes (Fig. 4, for example). Comparisons of bacteroid-pellet volumes following nodule tissue breakage and envelope rupture indicated that at least two thirds of the envelopes were destroyed at tissue breakage. Nevertheless, yields of intact envelopes were sufficient for specific spectrophotometrical analysis of leghaemoglobin and for the analysis of other components of envelope extracts. In 30-d-old nodules, on a whole-cell basis, leghaemoglobin is 0.7 mM (Ber- gersen and Goodchild 1973, Table 6) and 46% of the space in those cells is between the membrane envelope and the bacteroid surfaces and 17% is host cytoplasm (1.c., Fig. 8). In the envelope space the average concentration of leghaemoglobin was estimated to be 342 gM (Table 2). It can therefore be calculated that the concentration in the cytoplasm between the envelopes could be of the order of 3 raM. Partially oxygenated leghaemoglobin in the cytoplasm would thus represent a considerable store of oxygen in equilibrium with a low concentration of free O2, whilst the concentration of leghaemoglobin in the envelope space, although only one tenth as great, would be quite adequate for facilitation of 02 flux to the bacteroids (Bergersen 1980, 1981).

It was surprising that all of the leghaemoglobin components present in total nodule extracts (Fuchs- man and Appleby 1979) were present in envelope extracts in similar proportions (Table 3). There must be considerable exchange of macromolecules between the envelope space and the cytoplasm, where leghaemoglobin is synthesized (Verma and Bal 1976). Fur- ther, this exchange must occur in mature nodules (those used were 30 d old) since the ratios of leghaemoglobins change during development (Fuchsman and Appleby 1979). The possibility that the leghaemoglobin within the envelope space resulted from envelopes opening during the tissue breakage, then resealing, thus enclosing cytoplasmic components in- cluding leghaemoglobin, has been eliminated on sev-

eral grounds. Apart from the fact that no such events were observed microscopically, there were consistent differences between components present in the $1 nodule extract and the envelope extract. Envelope extracts contained an additional minor leghaemoglobin (Fig. 6b), and the "cathode band" seen when envelope extracts were subject to isoelectric focusing, was not detected in $1 preparations. Also, there were four to six times greater concentrations of fluorescent materials (Fig. 7) in envelope extracts than in $1 preparations (data not shown). Further, the results of experiments in which radioactively labelled proteins were present during tissue breakage and washing of envelopes, showed that no detectable exogenous proteins became occluded within the space from which the leghaemoglobin was released when the envelopes were ruptured. These results support the conclusion that the envelope space is a genuine compartment of nodule cells, whose contents are released when envelope preparations are ruptured by osmotic shock and centrifugal packing.

The electron micrograph of Fig. 1 was prepared by Celia Miller. Dr. S.W. Thorne kindly prepared the spectra of Fig. 7.

References

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tion and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules. J. Cell Sci. 30, 151-174

Schwinghamer, E.A., Dudman, W.F. (1980) Methods for identify- ing strains of diazotrophs. In : Methods for evaluating biological nitrogen fixation, pp. 337-365, Bergersen, F.J., ed. John Wiley, Chichester, U.K.

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Received 26 February; accepted 8 June 1981

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Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules