Abstract. The localization of H+-ATPases in soybean(Glycine max L. cv. Stevens) nodules was investigatedusing antibodies against both P-type and V-type en-zymes. Immunoblots of peribacteroid membrane (PBM)proteins using antibodies against tobacco and Arabi-dopsis H+-ATPases detected a single immunoreactiveband at approximately 100 kDa. These antibodies rec-ognized a protein of similar relative molecular mass inthe crude microsomal fraction from soybean nodulesand uninoculated roots. The amount of this protein wasgreater in PBM from mature nodules than in youngernodules. Immunolocalization of P-type ATPases usingsilver enhancement of colloidal-gold labelling at thelight-microscopy level showed signal distributed aroundthe periphery of non-infected cells in both the nodulecortex and nodule parenchyma. In the central nitrogen-®xing zone of the nodule, staining was present in boththe infected and uninfected cells. Examination of nodulesections using confocal microscopy and ¯uorescencestaining showed an immuno¯uorescent signal clearlyvisible around the periphery of individual symbiosomeswhich appeared as vesicles distributed throughout theinfected cells of the central zone. Electron-microscopicexamination of immunogold-labelled sections showsthat P-type ATPase antigens were present on the PBMof both newly formed, single-bacteroid symbiosomesjust released from infection threads, and on the PBM ofmature symbiosomes containing two to four bacteroids.Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect thisprotein on symbiosome membranes. Only a very faintsignal with this antibody was detected on Western blotsof puri®ed PBM. During nodule development, fusion ofsmall symbiosomes to form larger ones containing

multiple bacteroids was observed. Fusion was precededby the formation of cone-like extensions of the PBM,allowing the membrane to make contact with theadjoining membrane of another symbiosome. We con-clude that the major H+-ATPase on the PBM ofsoybean is a P-type enzyme with homology to othersuch enzymes in plants. In vivo, this enzyme is likely toplay a critical role in the regulation of nutrient exchangebetween legume and bacteroids.

Key words: ATPase ± Glycine max ± Nitrogen ®xation ±Symbiosome

Introduction

Nitrogen-®xing bacteroids within legume nodules aresurrounded by a membrane of plant origin ± theperibacteroid membrane (PBM) ± which e�ectivelyexcludes the bacteroid from the host cytosol and whichcontrols the movement of metabolites between the twosymbiotic partners (Whitehead and Day 1997). ThePBM and its enclosed bacteroids together make upthe symbiosome, the basic nitrogen-®xing unit of thenodule infected cell (Roth et al. 1988). The essentialmetabolic exchange between bacteroid and plant isreduced carbon into the bacteroid for ®xed nitrogen tothe plant. There is now general agreement that dicar-boxylates are the major source of carbon supplied to thebacteroid and that ammonium is the form in which ®xednitrogen is supplied to the plant (Day and Copeland1991; Vance and Heichel 1991; Day et al. 1995; Streeter1995; Whitehead et al. 1995). It addition to carbon/nitrogen exchange, many other metabolites and ions alsomust cross the peribacteroid membrane (Udvardi andDay 1997).

A major feature of the PBM is its energization by anH+-pumping ATPase. This was one of the ®rst enzymeactivities to be detected on the PBM (Robertson et al.1978; Verma et al. 1978) and biochemical studies on

*Present address: Timiryasev Institute of Plant physiology, RussianAcademy of Science, Botanicheskaya 35, Moscow 127276, RussiaAbbreviation: PBM = peribacteroid membrane

Correspondence to: D.A. Day;E-mail: david.day@anu.edu.au; Fax: 61 (2) 6249 0313

Planta (1999) 209: 25±32

Localization of H+-ATPases in soybean root nodules

Elena Fedorova1*, Rowena Thomson1, Lynne F. Whitehead1, Oliver Maudoux2, Michael K. Udvardi1, David A. Day1

1Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia2Unite de Biochimie Physiologique, University of Louvain, Croix du Sud, 2-20B-1348, Louvain-la-Neuve, Belgium

Received: 25 November 1998 /Accepted: 6 January 1999

soybean (Blumwald et al. 1985; Bassarab et al. 1986;Udvardi and Day 1989), lupin (Domigan et al. 1988)and pea (Szafran and Haaker 1995) indicate that theATPase most closely resembles a P-type ATPase. It isMg2+-dependent, has an acidic pH optimum, is at leastpartially inhibited by vanadate, and is stimulated bycations, particularly K+ and NH�4 . Studies with intactsymbiosomes have shown that the ATPase pumpsprotons and forms both a membrane potential (positiveinside) and a pH gradient (more acidic inside thesymbiosome; Blumwald et al. 1985; Udvardi and Day1989; Udvardi et al. 1991; Dubrovo et al. 1992; Szafranand Haaker 1995). This energization of the PBM has thepotential to profoundly a�ect the movement of otherions across the PBM and bacteroid membranes (Udvardiet al. 1991; Day et al. 1995; Tyerman et al. 1995; Corzoet al. 1997).

In the only molecular study of PBM ATPase to date,Campos et al. (1996) isolated a cDNA encoding a P-type

ATPase from Phaseolus nodules, but immunolocaliza-tion and RNA hybridization suggested that this proteinwas localized in the uninfected cells of the nodule. Theseauthors concluded that the PBM did not possesssigni®cant P-type ATPase. In this context, it should benoted that other types of ATPase activity have beenreported on the PBM, including V-type enzymes and Ca-ATPases (Blumwald et al. 1985; Bassarab et al. 1986;Dubrovo et al. 1992).

Here, a reinvestigation of the localization of H+

ATPases in soybean, using antibodies against bothP-type and V-type enzymes and nodules of di�erentages, is reported. Evidence is presented that in soybeannodules the infected cells contain appreciable quantitiesof P-type ATPases and that much of this is localized onthe PBM.

Materials and methods

Plants. Soybean (Glycine max L. cv. Stevens) seeds were inoculatedwith Bradyrhizobium japonicum USDA 110 and grown in anaturally illuminated greenhouse as previously described (Priceet al. 1987). Nodules were collected from plants at two develop-mental stages: young nodules (2±3 mm diameter) were harvestedfrom 10- to 14-d-old plants and mature nodules at 8 weeks afterplanting.

Membrane isolation and immunoblotting. Peribacteroid membranewas isolated from symbiosomes puri®ed from soybean nodules asdescribed by Day et al. (1989). Isolated PBM was subjected tophenol extraction to remove lipid from the proteins, using themethod of Hurkman and Tanaka (1986). Equal aliquots of PBMproteins were solubilized in sample bu�er [4% (w/v) SDS, 125 mMTris-HCl (pH 6.8), 10% (v/v) glycerol, 0.002% (w/v) bromophenolblue and 5% (v/v) mercaptoethanol] and kept at room temperaturefor 2 h. The proteins were then subjected to SDS-PAGE asdescribed by Day et al. (1989). A modi®ed version of the method ofTowbin et al. (1979) was employed for immunoblotting, usingantibodies to P-type and V-type ATPases. The following were used:polyclonal antibodies against the small loop (Glu 118 to Gln241) ofthe plasma membrane H+-ATPase from Nicotiana plumbaginifolia(isoform PMA2: Morsomme et al. 1996) expressed in Escherichiacoli as a fusion protein with glutathione S-transferase; polyclonalantibodies against the central domain of Arabidopsis thalianaplasma membrane H+-ATPase (isoform AHA3; Campos et al.1996); and monoclonal antibodies to the 70-kDa and 60-kDasubunits of vacuolar H+-ATPase from oat roots (7A5 and 2E7,respectively; Ward et al. 1992). Immunoreactive proteins werevisualized using the BM Chemiluminescence system (BoehringerMannheim, Mannheim, Germany) according to the manufacturer'sinstructions.

Fig. 1A±C. Immunoblots of P- and V-type ATPases in membraneextracts from soybean nodules. The various membrane fractions,prepared as described in Materials and methods, were separated bySDS-PAGE, blotted onto nitrocellulose membrane and probed withantibodies against the tobacco P-type ATPase or the 70-kDa subunitof the oat V-type H+-ATPase. In A and B, 8-week-old plants wereused. A P-type antibody was used. Lane 1, 10 lg PBM proteins; lane2, 10 lg root microsomal proteins. B V-type antibody was used. Lane1, 10 lg PBM proteins; lane 2, 10 lg crude nodule microsomalproteins; lane 3, 10 lg crude root microsomal proteins. C A singleWestern blot was cut into strips and probed with the di�erent ATPaseantibodies. Upper panel, P-type ATPase antibody; lower panel, V-typeATPase antibody. Lane 1, 15 lg microsomes from uninoculated rootsof 4-week-old plants; lane 2, 10 lg microsomes from 4-week-oldinoculated roots from which the nodules had been removed; lane 3,10 lg microsomes from 2-week-old uninoculated roots; lane 4 10 lgmicrosomes from 4-week-old nodules; lane 5, 10 lg microsomes from2-week-old nodules; lane 6, 20 lg PBM from 4-week-old nodules

Fig. 2A,B. Phase-contrast micrographs of sections from soybeannodules after immunolabelling with silver enhancement. A Controlsection treated with secondary antibody only. B Section treated withprimary antibody against P-type ATPase PMA2 for tobacco. IC,infected cell; VB, vascular bundle. Arrows point to deposition of labelin infected cells and around the periphery of uninfected cells.C Confocal microscopy with sections treated with primary antibodyagainst P-type ATPase PMA2 from tobacco. Immuno¯uorescentsignal is visible around the periphery of individual symbiosomes(arrow), which appear as vesicles distributed throughout the infectedcells. UN, uninfected cell IC, infected cell

c

26 E. Fedorova et al.: Peribacteroid membrane ATPase

E. Fedorova et al.: Peribacteroid membrane ATPase 27

Crude microsomal fractions were prepared from roots andnodules by grinding fresh tissue on ice in an equal mass ofhomogenization bu�er [350 mM mannitol, 25 mM Mes-KOH (pH7.0), 10 mM EGTA, 10 mM MgSO4, 5 mM dithiothreitol, 1 mMphenylmethylsulfonyl¯uoride and 1 lM E64], removing cellulardebris with ®ltration through Miracloth, followed by centrifugationfor 15 min at 27,000 g. The supernatant was then centrifuged at100,000 g for 1 h, followed by resuspension as above.

Electron microscopy. Sample preparation. For electron microscopywith immunolabelling, nodules were ®xed at 4 °C in 4% depoly-merized paraformaldehyde, mixed with 0.1% or 0.5% glutaralde-hyde in 0.05 mM potassium phosphate bu�er (pH 7.3), embeddedin Lowicryl (Sigma) at )20 °C, and polymerized under UV light.Alternatively, a standard ®xation in a 4% paraformaldehyde/3%glutaraldehyde mix was used, with post-®xation in osmiumtetroxide. This material was embedded in LR White resin (Sigma)and polymerized at 60 °C. Sections 70±100 nm thick were cut withan LKB ultratome with glass knives. Sections were mounted onto100- or 50-mesh gold or nickel grids or slots and processed forimmunolocalization, prior to observation with a JEOL-2000transmission electron microscope.

Immunolabelling. Post-embedding immunolabelling was performedaccording to standard protocols. Grids were incubated in phos-phate-bu�ered saline (PBS; 10 mM K-phosphate, 150 mM NaCl,pH 7.4) and double-blocked with 5% fetal albumin (Sigma) and3% normal goat serum (Sigma). Blocked grids were treated withprimary antibody diluted 1:80 (PMA2), 1:250 (AHA3) and 1:1(monoclonal 2E7) overnight at 4 °C, washed six times with PBS,and exposed to 12-nm colloidal gold conjugated with goat anti-rabbit or anti-mouse IgG1: 40 (Pierce), for 1 h at room temper-ature. Grids were then stained using 2% uranile acetate andReynold's lead citrate.

For immunolabelling with silver enhancement, 1-lm sectionswere cut and mounted on SuperFrost*/Plus microscope slides(Menzel-Glaser, Germany). Slides were treated with primaryantibody at the same dilution as for the electron-microscopylabelling procedure, followed by incubation in 6-nm colloidal goldconjugate with goat anti-rabbit, or anti-mouse Ig 1:30 (Pierce). Thesignal was enhanced with Silver Enhancing Kit (ICN, Irvine, Calif.,USA). As an additional control for immunolabelling speci®city,anti-Nodulin 26 antibody was used at a dilution of 1:400.

Confocal microscopy. Nodules were hand-cut into approximately60- to 70-lm slices by razor blade and ®xed in 1% paraformal-dehyde for 30 min at room temperature, washed six times withPBS, and left overnight at 4 °C in the solution of primary antibody(diluted 1:150 for the polyclonal PMA2). Auto¯uorescence wasquenched by incubating in 10% of NH4Cl for 10 min. A secondaryantibody-FITC conjugate (Amersham) was used at a dilution of1:25, for detection.

Confocal microscopy was conducted on an MRC-600 Confocalimaging system with a krypton-argon laser and a Zeiss Axiovert 10inverted microscope. Images collected electronically were processedby using Adobe Photoshop 3.

Results

Localization of P-type ATPase

Immunoblots. Phenol extraction of PBM was found toremove interfering lipids, leading to much better sepa-ration of PBM proteins during SDS-PAGE (and two-dimensional PAGE) and, consequently, better resolutionof separated proteins following Coomassie Blue or silverstaining. Accordingly, phenol-extracted protein wasused in all of the Western blots. Immunoblots of PBM

proteins using the PMA2 antibody against the NicotianaH+-ATPase (Morsomme et al. 1996) resulted in a singleimmunoreactive band at approximately 100 kDa(Fig. 1). Identical results were obtained with the anti-body raised against the Arabidopsis protein (results notshown). These antibodies recognized a protein of similarrelative molecular mass in the crude microsomal fractionfrom uninoculated soybean roots, inoculated roots fromwhich nodules had been removed, and in the crudemicrosomal fraction from nodules (Fig. 1C, lanes 1±5).The results indicate that the PBM contains a P-typeATPase similar to that found in root membranes. Theamount of this protein in microsomal preparations from2-week-old nodules was less than that from 4-week-oldnodules (Fig. 1C), but more reactive protein wasobserved in the PBM from 4-week-old nodules thanfrom 8-week-old nodules (Fig. 1A vs. Fig. 1C). In someblots the band on the PBM appeared to have a di�erentmobility on the gel than that in the microsomal fraction(Fig. 1A).

Light microscopy. Silver enhancement of colloidal goldlabelling allowed visualization of H+-ATPase with bothof the polyclonal antibodies (AHA3 or PMA2) at thelight-microscopy level (Fig. 2B). The silver grains rep-resenting the signal were found in rows along theperiphery of the non-infected cells in both nodule cortexand nodule parenchyma (Fig. 2B, arrow). Labelling wasalso observed over the phloem elements in the vascularbundles. This pattern of staining in consistent with a P-type ATPase localized on the plasma membrane of thesecells. In the central nitrogen-®xing zone of the nodule,staining was present in both infected and uninfectedcells. In the uninfected cells, silver granules weredistributed mainly at the periphery of the cell, againpresumably re¯ecting plasmalemma ATPase. In infectedcells, on the other hand, the signal was found through-out the cell (Fig. 2B), indicating the presence of epitopeson the symbiosomes.

Examination of nodule sections using confocal mi-croscopy and ¯uorescence staining with antibodiescon®rmed the results described above. In the infectedcells of the central zone, an immuno¯uorescent signalwas clearly visible around the periphery of individualsymbiosomes which appear as vesicles distributedthroughout the cell (Fig. 2C). In contrast to this, theonly obvious ¯uorescence in the uninfected cells was inthe plasmalemma region (Fig. 2C).

Electron microscopy. Electron-microscope examinationof immunogold-labelled sections showed that P-typeATPase antigens were present on the PBM of both

Fig. 3A±D. Electron micrographs of soybean nodule tissue treatedwith primary antibodies against P-type ATPases.A, B Sections treatedwith antibody against PMA2 from tobacco. Note the labelling overPBM and plasmalemma; C, D Sections treated with antibody againstArabidopsisAHA3 ATPase. Note fusion between two symbiosomes atthe possible late fusion pore stage in D (indicated by doublearrowhead). B, bacteroid; PBM, peribacteroid membrane.Bars = 200 nm

c

28 E. Fedorova et al.: Peribacteroid membrane ATPase

E. Fedorova et al.: Peribacteroid membrane ATPase 29

30 E. Fedorova et al.: Peribacteroid membrane ATPase

newly formed, single-bacteroid symbiosomes just re-leased from infection threads (Fig. 3A,C), and on thePBM of mature symbiosomes containing two to fourbacteroids (Fig. 3B,D). In addition, gold particles wereobserved consistently on endoplasmic-reticulum cister-nae and Golgi vesicles in infected and uninfected cells(not shown). Although gold particles were occasionallypresent over the bacteroid cytosol in sections treatedwith AHA3 Ab, this was considered to be non-speci®cbackground.

In nodules of this age (about 2 weeks after inocu-lation), fusion of small symbiosomes to form a largerone was observed frequently (Fig. 3D; Fig. 4A±D).Fusion was preceded by the formation of ®nger-likeextensions of the PBM, allowing the membrane tomake contact with the adjoining membrane of anothersymbiosome (Fig. 4A±C). These membrane appendagesresembled the ``sharp or gently curved corner'' de-scribed during membrane fusion events in mousekidney tubule cells (Fujioka et al. 1990). In the regionof the fusing PBM, gold particles were seen in the PBS(Fig. 3D), presumably re¯ecting release of the ATPasefrom the membrane as it degraded at the point offusion.

Localization of V-type ATPase

Monoclonal antibodies raised against subunits of thevacuolar H+-ATPase from oat roots, which have beenshown to cross-react with similar enzymes in otherplants (Ward et al. 1992), were used to investigate thedistribution of this enzyme in soybean nodules. Al-though immunogold labelling using antibody against the70-kDa B-subunit showed some reaction with otherendomembranes in infected cells, no signals was detectedon the symbiosomes (results not shown). In Westernblots, strong reactive protein bands were seen with theantibodies against the 70-kDa subunit in microsomalfractions from both nodules and roots (Fig. 1B,C); littledi�erence in expression was observed with plant age.Similar results were obtained with the antibody againstthe 60-kDa subunit (not shown). In some preparationsof PBM, a very weak reaction was seen with the 70-kDasubunit antibody (Fig. 1B, lane 1); in other preparations,no appreciable reaction was seen in PBM fractions (Fig.1C, lane 6). The antibody against the 60-kDa subunitreacted strongly with bacteroid proteins and was notused in immunogold labelling experiments (not shown).We conclude that the PBM does not contain appreciableV-type ATPase proteins, in agreement with biochemicalstudies (Ou Yang and Day 1992).

Discussion

The results presented here con®rm biochemical studieswhich have demonstrated P-type H+-ATPase activityon isolated symbiosomes and PBM (see Introduction).This ATPase is a reversible H+ pump which generates aDw and a DpH across the PBM (Udvardi et al. 1991;Szafran and Haaker 1995). Energization of the PBM islikely to play a crucial role in the regulation ofmetabolite transport across the PBM, particularly thecoordination and facilitation of malate and ammoniummovement between plant and bacteroid (Day et al. 1995;Udvardi and Day 1997). Our immunological data,obtained with antibodies against di�erent plantATPases, clearly show that the PBM contains a P-typeenzyme similar to that found on the plasmalemma. Insome Western blots, slightly di�erent-sized bands wereobserved between the PBM and root microsomalfraction (Fig. 1A), perhaps indicating that the PBMenzyme is a distinct isoform of the ATPase family.

Using the light microscope, high levels of immuno-labelling of P-type ATPase(s) were found in all nodulecells (Fig. 2). Label was found on the periphery of thecell in addition to the signal on the PBM. In contrast,labelling of the nodule with anti-nodulin 26 antibodyresulted in signal on the PBM only (not shown). Animportant aspect of plasmalemma H+-ATPase regula-tion is its activation by auxin in elongating tissues (Friaset al. 1996), where auxin-induced wall acidi®cation is anessential component of the elongation response andappears to be mediated by increased activity of theplasmalemma of the H+-ATPase (Evans 1985; Serrano1989; Rayle and Cleland 1992). The infected cells in thenodules we examined were undergoing substantialenlargement and it is possible that the plasmalemmaATPase of these cells is important for this process.

The cytosol of nodule infected cells in enriched withendoplasmic-reticulum cisternae and Golgi bodies, andthe tra�cking of vesicles is also intensive, especially inyoung nodules, due to proliferation of the membranesystem that generates components of the PBM (Roth andStacey 1989). It is possible that the ATPase detected hereplays a role in vesicle fusion with the symbiosome, andpossibly in the fusion seen between adjacent symbio-somes (Figs. 3, 4). To our knowledge, this is the ®rstreport of such fusion in young developing nodules andwas observed frequently. The multi-bacteroid symbio-somes which predominate in mature nodules of soybeanmay result at least partly from fusion of smallersymbiosomes. Merging of adjacent PBMs has beenobserved also in Phaseolus vulgaris nodules immediatelyprior to senescence (Pladys and Rigaud 1988).

In contrast to the results obtained with the antibodiesagainst P-type ATPases proteins of V-type enzymes werebarely detectable on the PBM. This is consistent with theinhibitor-sensitivity of the PBM ATPase (Domiganet al. 1988; Udvardi and Day 1989) and the pH pro®leof ATP-dependent energisation of the PBM (Ou Yangand Day 1992). Previous reports of V-type enzymes onthe PBM may have resulted from contamination withother cell membranes.

Fig. 4A±D. Electron micrograph of symbiosomes showing fusion ofPBM. A±C Cone-like membrane structures facilitate close contactbetween two membranes. D Complete fusion has occurred betweenadjacent symbiosomes. Double arrowheads, points of membranecontact and fusion; B, bacteroid; PBM, peribacteroid membrane.Bars = 200 nm

b

E. Fedorova et al.: Peribacteroid membrane ATPase 31

Conclusion

It is concluded that the major H+-ATPase on the PBMof soybean is a P-type enzyme with homology to those inother plants. In vivo, this enzyme is likely to play acritical role in the regulation of nutrient exchangebetween legume and bacteroids.

This work was supported by grants from the Australian ResearchCouncil to D.A.D. andM.K.U. The authors thank Dr. O. Schwartzfor the help with confocal microscopy, Prof. A. Hardham and Prof.F. Bergersen for discussion of the data, Prof. Serrano and Prof. M.Boutry for providing AHA3 and PMA2 Ab, Prof. H. Sze for 2E7and 7A5 antibodies, and Dr D. Roberts for Nod-26 Ab. R. Thom-son was the recipient of an Australian Postgraduate Award.

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