Plant and Soil 191: 189–203, 1997. 189c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Regulation of nitrogen fixation in infected cells of leguminous root nodules inrelation to O2 supply

F.J. BergersenDivision of Biochemistry and Molecular Biology, School of Life Sciences, Australian National University,Canberra, ACT 0200, Australia

Received 9 April 1997. Accepted 9 April 1997

Key words: bacteroids, leghaemoglobin, mitochondria, nitrogenase, oxygen, pro-plastids, regulation, respiration

Abstract

Respiration and nitrogen fixation in legume root nodules is considered to be limited by the rate at which O2 from theatmosphere can enter nodules. A thin diffusion barrier in the inner cortex, restricts access to the central tissue wherethere is a high demand for and low concentration of O2. Observed variations in rates of nodule activities in responseto imposed stresses, are often attributed to variations in the diffusion resistance of the barrier. In the present work,alternative or supplementary metabolic mechanisms are considered. Aspects of nodule structure and of metabolismunderlying nodule activities are reviewed in terms of components of the symbiotic system, the nature of steadystates and in relation to homeostasis of low concentration of O2 within the bacteroid-filled host cells. It is suggestedthat variations in O2-demand of both mitochondria and bacteroids, serve to preserve nitrogenase activity by poisingO2 concentration within ‘safe’ limits. Further, data from isolated soybean bacteroids suggest that nitrogenase isconverted to a less active but more robust form, in the presence of O2 in excess of about 70 nM, thus protectingnitrogenase from irreversible inactivation by excess O2. This regulation is rapidly-reversible when O2 concentrationfalls below about 0.1 �M. Respiration by large numbers of host mitochondria in the periphery of infected nodulecells, adjacent to gas-filled intercellular spaces, is considered to play an important part in maintaining a steepgradient of O2 concentration in this zone. Also, it is possible that variations in nodule O2 demand may be involvedin the apparent variations in resistance of the diffusion barrier. It is concluded that there are many biochemicalcomponents which should be considered, along with possible changes to the diffusion barrier, when the effects ofimposed stresses on nodule activities are being analysed.

Introduction

The root nodules of leguminous plants are capable offixing sufficient atmospheric N2 to meet the require-ments for combined nitrogen of rapidly-growing crop(e.g. Bergersen et al., 1992), forage (Gault et al., 1995)or pasture plants (e.g. Ledgard and Steele, 1992),whilst at the same time often contributing significantquantities also to the soil in which they are grown(e.g. Evans et al., 1987; Gault et al., 1995). To sus-tain N2 fixation and bring maximum benefit to thehost plant, the cells of the nodules and the symbioticbacteria within them must be supplied efficiently withenergy-yielding substrates generated by host photosyn-thesis. Since bacteroids have no fermentative capacity

and products of host plant fermentation are inhibitoryfor plant metabolism, the electron transport systemsof both host and symbiont must be unrestricted andtheir terminal oxidases supplied with sufficient O2 tosustain efficient generation of ATP; however, the O2-labile nitrogenase system of the bacteroids must notbe exposed to excess O2 (Robson and Postgate, 1980).Therefore, it is plain that O2 supply to root nodules isregulated to provide for adequate fluxes at low con-centrations of O2 ([O2] ) dissolved in the cytoplasm ofinfected nodule cells.

The following features of such regulation are gen-erally agreed: (i) The infected cells of nodule cen-tral tissue constitute a compact, intense sink for O2

(increased numbers of mitochondria compared with

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other plant tissues and thousands of bacteroids percell). (ii) Access of O2 from the surrounding atmo-sphere is restricted by a thin layer of cells containingfew gas-filled intercellular spaces, in the inner cortexof nodules, the diffusion barrier (Minchin and Wit-ty, 1990) or boundary layer (Parsons and Day, 1990).Thus, a region of low [O2], often below the detectionlimit of O2 microelectrodes, is generated in the centraltissue (Tjepkema and Yocum,1974; Witty et al., 1987).(iii) Under these conditions of restricted O2-supply, theflux of O2 to bacteroids respiring within symbiosomes,is enhanced (facilitated) by the presence of high con-centrations of the O2-binding, myoglobin-like haemo-protein, leghaemoglobin, located in the cytosol of thehost cells in which these symbiosomes are embed-ded (Appleby, 1984, 1994). Thus, the nodule systemcomplies with the general principal that all cytoplas-mic haemoglobin and myoglobin systems "operate insteady states of myoglobin partial oxygenation main-tained by rapid oxygen consumption operating in theface of a barrier to oxygen entry" (Wittenberg and Wit-tenberg, 1990).

The respiration and nitrogenase activities of nod-ules may decline quickly following the imposition ofcertain stresses (e.g. substitution of acetylene/O2 orArgon/O2 mixtures for air; Witty et al., 1984); thesedeclines are reversed by increasing the pO2 of the atmo-sphere. Such effects have been attributed to changesin the permeability of the nodules to O2 and thus toa variable diffusion barrier (e.g. Dakora and Atkins,1989; Layzell et al., 1993). This concept assumesthat O2-demand is unaffected by the stress. Howev-er, since there are metabolic components to the inter-acting regulation of O2 consumption and N2 fixationin nodules (e.g. Kuzma et al., 1993), the concept ofa variable diffusion barrier is obviously a simplifica-tion. Streeter (1995) has proposed that the metabolicbasis of the effects of stress imposed by non-invasivetreatments such as exposure of nodules to argon/O2

atmospheres, is to be found in changes to the protonbalance in the symbiosome space, with consequenteffects on bacteroid metabolism; mitochondrial func-tion is also affected by such treatments. The purpose ofthis present essay is to explore some different aspectsof the metabolic regulation of O2 - related nodule activ-ities, many of which have been attributed principallyto the operation of a variable diffusion barrier.

Some important questions

Is there a steady state in metabolism of infected cells?

In an apparent steady state, biochemical reactions in allof the compartments of infected cells and the metaboliccomponents within each compartment are in dynamicequilibrium. Rates of each reaction are steady, or moreprobably, are oscillating within narrow limits. In nod-ules, inputs via phloem of energy-rich substrates fromhost photosynthetic sources, are transformed from(mainly) sucrose to dicarboxylates (malate/succinate),probably in uninfected cells of the central zone of soy-bean nodules (e.g. Streeter, 1991) and imported intoinfected cells via a dicarboxylate carrier (Li and Day,1991). In these cells, import of energy-sources is bal-anced by their oxidation to provide ATP and reductantsfor fixation of atmospheric N2 into NH3, ATP for itsassimilation and for transport of assimilates out of theinfected cells. During a steady state, if there is anexcess of supply of exogenous substrates of photosyn-thetic origin, these are often sequestered in reservematerials such as bacteroid poly-�-hydroxybutyrate(PHB; Bergersen and Turner, 1990b) and the metabolicbalance is preserved. Despite the low [O2] prevailingin the cytoplasm of infected cells, most of the ATPneeded is generated from aerobic respiration of hostmitochondria and symbiotic bacteria, so supply of O2

must be an important component of a steady state. Theoverall limiting factor may not be apparent until thebalance is disturbed by an external change but it isassumed that a principal limiting factor in steady statemust be the supply of O2 (e.g. Layzell et al., 1990).All other reactions in the infected cell having becomeadjusted to the O2 supply rate imposed by the diffusionbarrier in the inner cortex of the nodule (e.g. Weisz andSinclair, 1987a,b). Although the low [O2] in the cen-tral nodule tissue may be low enough to initiate plantfermentative metabolism (Peterson and LaRue, 1981),aldehydes and alcohols are substrates for microaero-bic bacteroid respiration (Peterson and LaRue, 1981;Bergersen and Turner, 1993) and thus will be removed.

Limitation by O2-supply rate alone may not alwaysapply in apparent steady states. For example, withsteady, low rates of supply of C4-dicarboxylates to bac-teroids, equilibrium may be maintained by oscillationsbetween O2-limitation and C-substrate-limitation, asendogenous reserves are mobilized in an apparentlyO2-regulated fashion (Bergersen and Turner, 1992).Thus, under conditions of limited O2 supply to bac-teroids, it seems to be very likely that several compo-

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Figure 1. Transmission electron micrograph of parts of an infected cell and a neighboring uninfected cell in the central tissue of a soybeannodule. Features shown are (i), thick wall of an infected cell; (u) thin wall of an uninfected cell, with large vacuoles (v); (is) intercellular space(gas-filled in vivo); (m) mitochondria; (a) amyloplasts; (s) symbiosomes; (b) bacteroids with prominent granules of PHB (p). The bar scaleindicates 3 �m.

nents of substrate utilization and respiration are beingvaried to achieve homeostasis of [O2] and an appar-ent steady state. It follows that it may not always benecessary to invoke a change in the nodule gaseousdiffusion barrier to explain stress-induced changes innodule activities.

Such considerations raise the possibility that thereis an O2-sensor which may be initiating changes in res-piratory rates of the bacteroids. For soybean bacteroidsit is proposed that variations in malate concentrationcould affect the balance between activities of NAD-and NADP-linked malic enzymes, thus balancing theTCA and PHB cycles (Day et al., 1994) and maintain-ing O2 demand, for example, in the face of decliningsupply of exogenous substrates.

Are structural considerations important?

The central tissue of many (but not all) legume rootnodules is composed of two types of transformedparenchymatous root cells, viz. vacuolate, uninfect-ed interstitial cells and the infected cells (which maybe vacuolate or non-vacuolate and contain hundreds oreven thousands of symbiotic bacteria - the bacteroids).The relative numbers of the two cell types may beaffected by growth conditions (Dakora and Atkins,1990). Amongst nodules whose central tissues con-tain no uninfected cells, those of lupin and peanut arewell-known exanples. The following descriptions arefor soybean, whose nodule central tissue contains bothcell types. The shape of the central-tissue cells as seenin sections, suggests that they are of polyhedral shape.Prominent gas-filled intercellular spaces are locatedat the edges of the faces of each of the cells (Berg-

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ersen and Goodchild, 1973) and these spaces form aninterconnected network throughout the central tissue(e.g.Bergersen, 1996). Thus, the central tissue consistsof aqueous and gas phases.

The infected cells (Figure 1) are multi-compartmented, containing many symbiosomes, themembrane-enclosed structures containing the bac-teroids (e.g. Bergersen, 1982). Symbiosome mem-branes ( or peribacteroid membranes, PBMs) havedistinct properties and unique transport systems, thusimparting to the symbiosome space unique characteris-tics within which the bacteroids must function (Udvar-di and Day, 1997; Whitehead and Day, 1997). In eachcompartment and cell type, many interacting metabol-ic systems are present (e.g. Atkins, 1991; Day andCopeland, 1991; Streeter, 1991). In some tissue com-partments, rates of diffusion of say, O2, N2, H2 or ofsubstrates for which there is no transport mechanism,across a boundary in which there is a change of phase(e.g. from a gas-filled space into an aqueous phaseor from an aqueous phase across a lipo-protein mem-brane), may not depend only upon the gradient of con-centration of the solute across the boundary. The ratesat which solutes dissolve from one unstirred phase intoanother and the partition coefficients for the solutesbetween the phases, may be more important.

The structural elements in infected cells have beendescribed in terms of their dimensions and numbers,the reactions occurring in or through them and the roleswhich O2 flux or concentration may play, directly orindirectly affect the total activity of the infected cell.Recent simulation models consider infected cells ofsoybean nodules (Bergersen, 1994, 1996; Thumfort,1996; Thumfort et al.,1994). The former author uses arhombic dodecahedron-shaped cell and includes newdata about the number and location of mitochondriaand calculations of N2fixation driven by bacteroid res-piration. This structural model will provide the frame-work for what follows but differences between soybeanand other nodule types will be mentioned as appropri-ate.

What is the role of O2 demand in distribution of O2

within cells?

Physiological consideration of diffusion of O2 intonodules frequently uses the Ohm’s law analogue inwhich O2 is distributed through a circuit containingvarious diffusion resistances through which the poten-tial is dropped from the [O2] of the external atmosphereto a very low value near the centre of infected cells.

Figure 2. The effects of two different O2 demands on O2 con-centration at a constant O2 flux and and constant affinity of theterminal; oxidase for O2. The curve (�) is for bacteroid respira-tion with 0.5 mM succinate (Vmax = 3.4�10�7 mols O2 s�1 (gdry wt.)�1 and (N) is for respiration with 0.5 mM malate (Vmax= 4.8�10�7mols O2 s�1 (g dry wt.)�1. For both the appKm(O2)= 21.5�10�5 mol m�3(Bergersen and Turner, 1993). The verticallines (A,B) indicate the two values of [O2] at which respiration is2�10�7 mols O2 s�1(g dry wt)�1

Such models are inadequate if they do not considerthe underlying metabolism, by attributing changes inO2-flux solely to changes in diffusion resistance. It isthe O2-demand of respiring organelles and bacteroids,which drives the generation of gradients of [O2] downwhich O2 diffuses within the various compartments ofthe cell. This property of respiration is illustrated inFigure 2, which shows how an increase in O2-demandcan drive the [O2] downward even when there is nochange to O2 supply conditions or to the flux of O2.The O2-demand, or the O2 sink strength, is best under-stood as being a function of the Vmax of a respiringbody.

When a nodulated root is exposed to an argon-O2 or an acetylene-O2 atmosphere, C-substrates usedhitherto for assimilation of fixed N, become availablefor other purposes. Such an increase would occur inthe cytosol surrounding symbiosomes and thus couldcontribute to an increased bacteroid O2-demand, as inFigure 2, causing a decline in [O2] in infected cells(e.g from A to B in Figure 2). Also, an increase in[C-substrate] would lead to diminished nitrogenaseactivity, because, after such a change, respiration sup-ports nitrogenase activity less efficiently (Bergersenand Turner, 1993; Bergersen, 1997). The decline innitrogenase activity could be reversed by a rise in exter-nal pO2. Although such a response does not fit all of theobserved physiological effects which are seen follow-ing acetylene/O2 or argon/O2 treatment (e.g. Witty et

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al., 1984), it seems probable that a change in bacteroidO2 demand is a component of the changes which areobserved following imposition of such treatments.

Other features of nodule structure and metabolism

Interfaces between infected cells and gas-filledintercellular spaces

Microscopy of fresh sections of nodules in air indi-cates that most, if not all of the intercellular spacesin the central tissue are gas-filled and interconnected(Bergersen and Goodchild, 1973a; Bergersen, unpub-lished; Sprent, 1972; Witty et al., 1987), whether thesespaces are between adjacent infected cells or at junc-tions with uninfected cells. In soybean nodules, gas-filled intercellular spaces in the central tissue have vari-ous dimensions. In chemically-fixed, Epon-embeddedsectioned tissue (Figure 1), the cross-sectional areasof the spaces were between 1 and 8 �m2, accordingto the age of the nodules and position in the tissue,occupied 1.5 to 5% of the tissue volume (Bergersenand Goodchild, 1973b; Dakora and Atkins, 1990)and the total areas of the interfaces amounted to 20to 34% of the surfaces of infected cells (Bergersenand Goodchild, 1973a; Brown and Walsh, 1994). Incontrast, van Cauwenberghe et al. (1993), using cry-oplaned frozen tissue reported somewhat smaller gas-filled spaces, with cross sections of 3 to 5 �m2/space,occupying < 1% of the central tissue volume. Thisresult led to estimates of only about 12.5% of the sur-face area as interfaces between infected cells and inter-cellular spaces (Thumfort et al., 1994). Microscopyof serial sections of embedded soybean nodules indi-cates that these spaces form an interconnected networkover the surfaces of infected cells. An estimate of thetotal length of the spaces in this network, based on1.5% of central tissue volume as intercellular spaceand an average cross section of intercellular spaces of3.9 �m2, yields the surprising total of 100 to 300 mper nodule. In any approximately median section ofan infected cell, there are 5 - 7 interfaces with spaces,suggesting a structure which has been modelled as arhombic dodecahedron, the intercellular spaces beinglocated along the edges of each rhombic face (Berg-ersen, 1994, 1995; Thumfort, 1996). The structure ofthis network investing an infected cell is illustrated inBergersen (1996). In this model, the total length ofintercellular space per infected cell is about 800 �mwith an interface between space and infected cells of

Table 1. The thickness of cell walls in the central tissue of soy-bean root nodules. Measurements made on transmission elec-tron micrographs of soybean (cv.Lincoln) nodules containingB.japonicum strain CB1809, aged 17 - 30 d. From materialdescribed by Bergersen and Goodchild (1973) and re sectionedin 1993

Interface Thickness (�m)a n

infected cell/intercellular space 0.37�0.031 12

uninfected cell/intercellular space 0.099�0.0003b 12

infected cell/infected cell:

< 3�m from the space 0.34�0.03 8

> 3 �m from the space 0.15�0.02 6

a From protoplast to outer cell wall surfaceb Walls of uninfected cells were uniform irrespective of interfaces.

4 �m average width. Other cell geometries have beenproposed, e.g. Thumfort et al. (1994) proposed a cubiccell structure, with the intercellular spaces at the edgesof each square face; such a structure does not accordwith the microscopic observations outlined above butthe authors then considered it to be a sufficient approx-imation and was considered superior to the sphericalmodel cell used by Sheehy and Bergersen (1986); sub-sequently (Thumfort, 1996) a dodecahedral model wasalso considered.

By transmission electron microscopy of thin sec-tions of soybean nodules (Figure 1), the cell walls ofinfected cells beneath intercellular spaces are usuallythicker than elsewhere in the central tissue (Table 1)and the surface of the wall in the space is seen asa thin layer of increased electron density (stained byOs, Pb, U), continuous with the middle lamella ofadjoining walls. There have been no specific studiesof the properties of the walls of nodule cells, but,when considering diffusion of O2 from the intercel-lular spaces into infected cells, it is necessary to notesome of the features found in other plant cell walls.There is no microscopically-detectable lignification ofwalls of infected cells of soybean, clover, pea or lupinnodules (author’s unpublished observations). Such pri-mary walls from other plant tissues are of variable andcomplex structure, composed of cellulose microfibrilsembedded in a watery matrix containing hemicellulos-es, pectins, glycoproteins, enzymes and other proteins(e.g. reviews by Bolwell, 1993; Cassab and Varner,1988; Northcote, 1972; Roland and Vian 1979). Thedisposition of water within the wall (Mackay et al.,1988), the hydrophobicity of dispersed componentsand the orientation of microfibrils in lamellar arraysparallel to the cell surface (e.g. Hogetso, 1986), all

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play important parts in determining the properties of aparticular wall. There have been few if any recent stud-ies of diffusion of O2 through walls, possibly becauseFrey-Wyssling (1976) stated authoritatively that pri-mary plant cell walls were "holopermeable", offeringno hindrance to the passage of micromolecular solutes.However, Canny (1990) has shown that diffusion of thefluorescent dye sulphorhodamine G (SR; mol.wt.553)through the wall apoplast of wheat leaf epidermal cellswas very much slower than expected (the diffusioncoefficient, DSR, was 4 - 8�10�12 m2 s�1, comparedwith an expected value of about 5�10�10 m2 s�1forSR in water at 25 �C). Since values of D for moleculesof up to 1000 daltons are inversely proportional tothe square roots of their molecular weights (Graham’sLaw; e.g. Vaupel, 1976), a value for DO2 in the range16.6 - 33�10�12 m2 s�1 can be calculated for thesewalls. Such a value is 0.01 to 0.05 of that expected formolecular-dispersed solutions of similar water content(Vaupel, 1976) and is probably a consequence of wallstructure and/or wall matrix properties. If this is so, rel-atively small changes in the walls of nodule cells, suchas the extrusion of a glycoprotein, as in walls of thenodule cortex (Ianetta et al., 1993; James et al., 1991)or a change in the matrix viscosity, may affect DO2 forthe cell wall profoundly, thus regulating entry of freeO2 into the infected cells of nodules. So far, there isno evidence for such regulation but with O2 consump-tion rates calculated for model cells (Bergersen, 1994)and the average of the above values for DO2 in cellwalls (25�10 �12 m2 s�1), the drop in concentrationof free, dissolved O2 across the wall interface with anintercellular space could be of the order of 40 �M.

Another aspect of the cell wall which will affectintracellular concentration of O2, is the nature of thesolution at the surface which faces the gas-filled space.If this solution contains a relatively high concentra-tion of macromolecular solutes thus reducing the wateractivity and increasing the viscosity by intermoleculareffects, the equlibrium concentration of O2 dissolvedin the surface of the wall may be lowered. Again, thereare no data about the nature of the surface solution inthis location but this possibility should be considered.

The symplast and apoplast of central tissue

From the earliest observations, electron microscopy ofnodule tissue revealed plasmodesmata connecting cellsof the central tissue and their role has been acknowl-edged by several authors (e.g. Pate et al., 1969; Selk-er and Newcomb, 1985). However, only recently has

the symplast been examined quantitatively. Brown etal. (1995) have shown that all of the central tissueforms a symplast but the frequency of plasmodes-mata is 2.5 times greater between infected cells anduninfected cells (0.023 �m�2 ) than between adja-cent infected cells (0.009 �m�2). Generally diameters(0.02 - 0.04 �m) of plasmodesmata are less than theirlength (0.08 - 0.24 �m), data which affect transportthrough them (Davis et al., 1987). In the young nod-ules examined by Brown et al. (1995), there were 22 to68 plasmodesmata connecting neighbouring infectedcells and 58 to 132 connecting an infected cell witheach adjoining uninfected cell. The small diameter ofplasmodesmata indicates a maximum total cross sec-tion area of only about 0.03 �m2 connecting infectedand uninfected cells. Properties of plasmodesmata inother locations have been reviewed by Wolf and Lucas(1994). Brown et al. (1995) concluded that there is nophysical restriction to transport of sucrose into nodulecells by symplastic or apoplastic routes.My own calcu-lations, based on the amount of sucrose needed to meetthe respiratory requirement of the model infected cell(Bergersen, 1996), and using the frequency and diame-ter of plasmodesmata reported by Brown et al. (1995),indicate that plasmadesmatal transport almost exactlymatches the respiration. However, this conclusion maynot apply to the transport of C4-dicarboxylates via thesame route. Other factors apply when consideration isgiven to entry of O2 into infected cells.

The concentrations of dissolved O2 in neighbour-ing infected cells are likely to be similar but the thinwalls of uninfected cells, their interfaces with gas-filled intercellular spaces and the comparatively loweroxygen demand of these cells (few mitochondria, e.g.Figure 7a in Bergersen, 1982; Newcomb et al., 1985)indicates a higher concentration of dissolved O2 thanin infected cells. (Indeed, this seems to be requiredfor the oxidation of uric acid to allantoin in the urei-de pathway; Atkins, 1991). Consequently transfer ofO2 from uninfected cells to infected cells via the sym-plast will occur. Also, there may be some transferfrom intercellular spaces via the cell wall apoplast,however , it seems likely that transfer via these tworoutes will be small compared with transfer direct-ly from the gas filled intercellular spaces. Transferof O2 from uninfected cells across the thin cell wallsinto infected cells may be a significant supplement tothat entering directly from the gas-filled intercellularspaces. However, this possibility has not been consid-ered in published simulations of infected cells (Berg-ersen, 1994, 1996; Thumfort et al., 1994). However,

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from a more recent simulation, Thumfort (1996) hasconcluded that it could account for up to 25% of theO2 entering infected cells.

Transport of O2 in the cytoplasm of infected cells

The cytoplasmic solution (cytosol) of an infected cellof soybean nodules, is a molecular dispersion, inwater, of soluble proteins (in soybeans ca. 18% w/v;Bergersen, 1993), including approximately 3 - 4 mMleghaemoglobin (Bergersen, 1982, 1994), the cytoso-lic enzymes (Atkins, 1991; Day and Copeland, 1991),other undefined macromolecules, micromolecules andions. The passage of O2 into the cytoplasm is consid-ered to be analagous to entry into a mammalian redblood cell (Nicholson and Roughton, 1951) or mus-cle cell (Wittenburg, 1970). That is, free O2 passesthrough the plasma membrane, which probably has alow resistance to diffusion, dissolving in the unstirredcytoplasmic solution within the cell. Near the cell sur-face this solution is different from the bulk cytoplasmdue to boundary effects. One of these effects is dueto the diffusion distance required to reach equilibri-um between O2 and leghaemoglobin (Lb). It has longbeen known that nodule Lb is concentrated in, if notconfined to the cytosol of infected cells, where it isin the Fe2+ form and is partially oxygenated (to formLbO2; reviews Appleby, 1984,1994) and facilitatesthe flux of O2 in the cytosol and may contribute toleghaemoglobin-mediated oxidative phosphorylationby mitochondria and bacteroids (Wittenberg and Wit-tenberg, 1990). The cellular concentration of Lb isincreased when nodules are grown at low pO2 (Dako-ra et al., 1991). Recent studies of the spectra of lightscattered from surfaces of undisturbed nodules in situ(King et al., 1988; Kuzma et al., 1993; Layzell et al.,1990) have indicated that, in unstressed soybean nod-ules grown in air, the concentration of free O2, ([O2])in equilibrium with Lb in the location being observed,was in the range 5 - 60 nM. Such values are derivedfrom estimation of the proportion (Y) of the total Lbwhich is in the oxygenated form (LbO2) and the equi-librium constant (K’) for O2-binding (K’ is the [O2] atwhich the Lb is 50% oxygenated; Y = 0.5). For Lbsfrom different legumes, K’ may be different (e.g. K’ =47 nM for soybean Lba; for Lb I from Pisum sativum,K’ = 149 nM; Appleby, 1984). Thus it will be apparentthat for equivalent oxygenation of Lbs, [O2] will bedifferent in pea and soybean nodules.

Facilitation of oxygen flux in cytoplasm is bestunderstood by considering that the total flux in a layer

of cytoplasm containing partially oxygenated Lb is thesum of the diffusive flux of free, dissolved O2 plus thediffusive flux of LbO2 (Wyman, 1966) and that LbO2,Lb and free dissolved O2 are in equilibrium at all pointsin the oxygen transport pathway from a region of high-er [O2] to a region of lower concentration (Wittenberg,1970). Since the concentration of Lb is large (sever-al mm) compared with [O2] (usually in the nm range),it will be apparent that the diffusion of LbO2 is dom-inant in the process. The flux (free O2) is a functionof dC and DO2 ( respectively the concentration gradi-ent and diffusion coefficient for free O2 dissolved incytoplasm). The LbO2flux is a function of dYCp andDLbO2 (where Cp is the [Lb], dY is the gradient of itsoxygenation and DLbO2 is the diffusion coefficent forLbO2 in cytoplasm; Bergersen, 1993). The delivery ofO2 to the surfaces of respiring mitochondria and sym-biosomes embedded in the cytosol (as distinct from thetransport of O2 through the cytosol) is governed by the[Lb], its relative oxygenation (Y) and the kinetics ofoxygenation (see above). In a model infected cell ofvolume 1.24�10�13 m3 (Bergersen, 1994) in which20% of the volume is occupied by cytoplasm contain-ing 2.79 mM Lb, with Y= 0.2, O2 will be dissociatingfrom LbO2at a rate of 7.6�10�14 moles s�1;(koff =5.5 s�1; Appleby, 1984). O2 consumption rates in themodel nodule cell are about 0.3 - 1.0�10�14 molesO2 s�1under the above conditions (Bergersen, 1996),so the rate of dissociation of LbO2 is > 7 times therespiration rate and thus cannot limit respiration.

The distribution of oxygen through the cytoplasmis impeded by structures lying within it and which arenot permeated by Lb. Such bodies reduce the volumeof cytoplasm through which oxygen is being transport-ed and they increase the tortuosity of the diffusion path.Close to the outside of infected cells lie the mitochon-dria and amyloplasts (Figure 1) which together occupyabout half of the volume of this zone in soybean cells(Bergersen, 1994; Millar et al., 1995). The distribu-tion of these organelles is similar in nodules of otherlegumes but they have not been studied quantitatively.

Beneath the mitochondrial zone, the cytoplasmcontains embedded symbiosomes (formerly known asperibacteroid units, PBUs; Figure 1), the stucturalunits, comprising the bacteroids enclosed within thesymbiosome (peribacteroid) membrane (PBM). Sym-biosomes are closely-packed and are partially in con-tact with each other. In infected cells of soybeannodules, it has been estimated that cytoplasm occu-pies only 15 to 20% of the available space (Berg-ersen and Goodchild, 1973b; Bergersen, 1982). Stud-

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er et al. (1992) challenged these intracellular propor-tions, claiming that high-pressure freezing techniquesimproved preservation of soybean nodule tissue forelectron microscopy and showed that the symbiosomemembrane is closely applied to the bacteroids, result-ing in less symbiosome space and therefore in rela-tively more cytoplasm. However, these authors usedonly very young nodules and no quantitative measure-ments were made of the distribution of cytoplasm. Inother types of nodules, often there is a large centralvacuole, thus reducing the number of symbiosomesper cell. However, in such cells the symbiosomes aresimilar in size to the soybean symbiosomes (althoughoften different in content, each containing, perhaps,only one much-enlarged bacteroid) and they occupya similar proportion of the cytoplasm (e.g. Figure 1bin Bergersen, 1982). As oxygen is distributed throughthe cytoplasm, to be used in bacteroid respiration, thegeometry of the distribution of cytoplasmic volumebetween the symbiosomes will have a strong effecton the gradients of concentration of O2 and LbO2; ifsymbiosomes touch, the diffusion path may be greatlylengthened. This attribute may be a powerful regu-lator of distribution of oxygen in infected cells, par-ticularly under the influence of changes in cell tur-gor (Durand et al., 1987; Pankhurst and Sprent 1975).Also, changes in cytoplasmic volume may alter con-centration of macromolecules and thus have an effecton DLbO2 (Bergersen, 1993).

The roles of mitochondria

It is well-known that the mitochondria of infectedcells are located in large numbers near the intercellu-lar space interfaces (e.g. Bergersen, 1982; Newcombet al., 1985; Figure 1). Recently, morphometric stud-ies have enabled quantitative description of the num-bers and distribution of mitochondria in these cells(Bergersen, 1994; Millar et al., 1995). There, the mainroles of mitochondria are to participate in the provisionof carbon skeletons and ATP for assimilation of NH3

fixed from N2 and to provide ATP for energization ofthe symbiosome membrane and thus for transport ofdicarboxylates into symbiosomes (Day and Copeland,1991; Streeter, 1991).

Mitochondria prepared from soybean nodules haverespiratory activity similar to those from roots (Dayand Mannix, 1988) and a role for leghaemoglobin inmicroaerobic respiration by nodule mitochondria hasbeen suggested (Rawsthorne and LaRue, 1986; Sug-anuma et al., 1987). Recently, Millar et al. (1995)

have shown that the cytochrome oxidase of mitochon-dria prepared from soybean nodules, has an appar-ent Km for O2 of 50 nM while deoxygenating LbO2;

with NADH or malate + glutamate as substrate; theVmax for the cytochrome pathway is 170 nmol O2

min�1(mg protein)�1. Mitochondria from soybeanroots and cotyledons are less well-adapted for respi-ration at low [O2], the app Km (O2) being 125 and146 nM respectively (Millar et al., 1994). These valuesfor Km and Vmaxof nodule mitochondria, when usedin infected cell simulation models (Bergersen, 1994,1996), indicated that at least half of the total cell respi-ration may be due to mitochondria and this proportionincreases as the cell experiences increasing availabili-ty of O2. Thus, the mitochondria, which are compactsinks for O2, play a significant ’protective’ role, con-suming O2 in the periphery of the infected cells andallowing the [O2] near the bacteroids to be maintainedat favorable levels (say <50 nM) for nitrogen fixa-tion, despite [O2] near the cell surface being as high as>2 �M (Bergersen, 1994). These findings supplementthe results from the cubic cell model (Thumfort et al.,1994) in which steep gradients of [O2] occurred in thezone of the cell now known to contain large numbersof mitochondria (Millar et al., 1995).

In cell-free preparations of nodule mitochondria,although the rate and efficiency of phosphorylationdeclined as the [O2] diminished in the microaerobicrange of interest, ATP production appeared to be suffi-cient to sustain NH3 assimilation (including the initialsteps of purine biosynthesis) and probably for transportof dicarboxylate into symbiosomes also (Millar et al.,1995). The apparent kinetics of mitochondrial respira-tion were altered when exogenous ADP was limiting,so that the O2 consumption was unaffected below butgreatly curtailed at [O2] above 50 nM (Millar et al.,1995). Thus, [ADP] becomes a powerful regulator ofmitochondrial O2 demand, when interacting with [O2]in the range calculated to prevail in the mitochondrialzone of the simulated infected cell (Bergersen, 1996).

In soybean nodules deprived of C-substrate (e.g.in darkness during pod-filling; Bergersen et al., 1991;Bergersen and Turner, 1992), when bacteroid respira-tion is being sustained by utilization of bacteroid PHB,mitochondrial respiration also may be C-substrate-limited, contributing to a possible rise in intracellu-lar [O2]. However, with resumption of photosynthesisin the shoots in daylight and transport of products tothe nodules, the location of the mitochondria near thecell surface and their high respiratory capacity, wouldbe expected to promote an early increase in oxygen-

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demand and thus contribute to lowering [O2] in theinfected cells to levels safe for nitrogenase. ShouldO2-supply to nodule central tissue become restrict-ed by operation of a variable diffusion barrier (seeabove), it is probable that in the mitochondrial zoneof infected cells, [O2] may fall to levels at whichATP production by mitochondria is restricted (Mil-lar et al., 1995). A restriction of the availability ofcytoplasmic ATP then could limit assimilation of NH3

fixed from N2 and/or transport of dicarboxlate sub-strates across the symbiosome membranes for sup-port of bacteroid activities (Day and Copeland, 1991).Either of these results would affect nitrogenase activi-ty indirectly. Therefore it seems reasonable to proposethat ‘nitrogenase-linked respiration’ of intact nodules(Minchin and Witty, 1990) may contain a significantcomponent of mitochondrial respiration.

Amyloplasts

These organelles (sometimes termed proplastidsbecause they do not always contain starch, despitebeing highly differentiated ) occur in close proximityto the mitochondria in infected cells of soybean nod-ules (Figure 1) and in this zone of the cells they occu-py about the same proportion (25%) of cytoplasmicspace as do mitochondria.Although amyloplasts do notthemselves consume O2, they are the sites of importantamino acid transformations (Robinson et al., 1994) andof the synthesis of inosine-5-monophosphate, a keyureide precursor, in the assimilation of NH3 fixed fromN2 in soybean and cowpea nodules (Atkins, 1991).These last reactions are important consumers of ATP,releasing ADP into the cytosol. Thus, amyloplasts con-tribute ADP in the mitochondrial environment. A suf-ficient supply of ADP is essential for the maintenanceof high rates of mitochondrial respiration (Millar et al.,1995). Therefore, the amyloplasts have an importantrole in stimulating mitochondrial respiration and thus,in maintaining a low [O2] in the outer layers of infectedcells. Recent studies have shown that both amyloplastsand mitochondria prepared from cowpea nodules, havea complete complement of the enzymes needed for denovo purine synthesis (Atkins et al., 1997) empha-sizing the close metabolic relationships between theseorganelles.

Symbiosome membranes and space.

Symbiosomes are membrane-enclosed packets ofendosymbiotic bacteria (bacteroids), embedded in the

cytoplasm of infected cells (Figure 1). They are usual-ly about 3 �m in diameter (Bergersen, 1982). (At thispoint it should be remembered that not all leguminousnodules contain symbiosomes; instead the microsym-bionts may be retained within tube structures: e.g. deFaria et al., 1987). Little is known of the diffusion ofO2 into or within symbiosomes. Since the symbiosomemembrane is similar in origin to the plasma membraneof infected cells (Robertson et al., 1978), but sharesfeatures with vacuolar membranes (Verma et al.,1993),it is reasonable to suppose that its permeability to O2

is similar. However, the symbiosome membrane is notpermeable to LbO2 and so only free, dissolved O2 maypass. As a result, there will be a thin boundary layer inthe cytoplasm outside the symbiosome, in which therewill be no Lb-facilitated O2flux . Essentially this layeris the thickness of solution through which LbO2 dif-fuses in the time taken for a molecule to release its O2

(koff = 5.6 s�1; Appleby, 1984). For a symbiosome ofdiameter 3 �m, in which bacteroids are consuming O2

as described by Bergersen (1994), the boundary layerwill be about 0.005 �m thick and it will only slight-ly increase the diffusion resistance of the symbiosomemembrane (Bergersen, 1996).

Within the symbiosome, there will be a gradient ofconcentration of dissolved O2 from the inner surfaceto the bacteroid surface, generated by the O2-demandof the bacteroid(s). This gradient, steepest near thesymbiosome membrane, will ensure that there is nonet diffusion of O2 out of symbiosomes. The steepnessof the gradient will be influenced by the magnitudeof the inward flux of O2, the distance between thesymbiosome membrane and the bacteroid surface(s)and the diffusion coefficient for O2 dissolved in thesymbiosome solution. In some legumes (e.g. clover,Bergersen, 1982; and alfalfa, Paau et al., 1980) thesymbiosome membrane is closely applied to the bac-teroid surface and there would be little difference in[O2] between them. However, in chemically-fixedsoybean nodules the space between bacteroid surfaceand symbiosome membrane appears much greater (e.g.Bergersen, 1982), although this may result from fix-ation technique (Studer et al., 1992). In chemically-fixed soybean nodules, about one half of the sym-biosome space is occupied by bacteroids (Bergersen,1982). Without leghaemoglobin within the symbio-some (Fuchsman, 1992), the [O2] restricts bacteroidrespiration to between 70 and 87% of respiration ofbacteroids respiring at the [O2] in the cytoplasm outsidethe symbiosome (Bergersen, 1996). Any mechanismaffecting the distribution of space within the symbio-

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some, such as small changes in cell turgor, could beexpected to have a significant effect upon the [O2] at thebacteroid surface. The solution within the symbiosomecontains several enzymes but the protein concentrationis probably much lower than in cytoplasm (Werner,1992). Therefore it is considered that the diffusioncoefficient for dissolved O2 within symbiosomes willbe similar to that in water. Apart from their as yetunknown permeability to O2, the symbiosome mem-branes exert profound influences on nodule activitiesparticularly by means of their selective permeability toenergy-yielding substrates supporting bacteroid activ-ities (e.g. Day and Copeland, 1991; Udvardi and Day,1997).

Bacteroids

In steady state, the respiration of bacteroids will beaccording to the kinetics of their terminal oxidase(s),providing that there is no other limitation such as limit-ing supply of energy-yielding substrate. Terminal oxi-dases of many bacteria appear to have a high affini-ty for O2: The development of experimental systemsbased on the deoxygenation of leghaemoglobin andother oxygen-carrying haemoproteins (Bergersen andTurner, 1979) has facilitated study of these oxidasesand in the case of root nodule symbioses, has been usedto study suspensions of bacteroids isolated from soy-bean, cowpea, sesbania (stem and root nodules), peaand several other legumes. At first, results suggestedthat some bacteroid terminal oxidases had unusuallyhigh affinity for O2 (e.g. Km = 6 � 4 nM O2 for soy-bean bacteroids; Bergersen and Turner, 1980, 1990a),with complex kinetics. Whilst bacteroids undoubted-ly are capable of respiring and fixing N2 at very low[O2], some of the results appear to have been dueto C-substrate limitation coming into effect at about20 nM O2, giving false values for apparent Vmax (O2)and Km (O2). Now it is considered that the princi-pal oxidase supporting N2 fixation by soybean bac-teroids has a Km(O2) of 20 to 29 nM (Bergersen andTurner, 1993), provided that measurements are madeduring steady states begining at least 20 min after achange in conditions. (For accounts of the possiblenature of these oxidases, see Hennecke, 1993; Preisiget al., 1993; Thony-Meyer et al.,1995). A variationin the substrate supply or in the rate of supply of O2,frequently initiates a change in bacteroid O2-demand,disturbing relationships beween [O2] and rate of respi-ration; sometimes oscillations are induced (Bergersenand Turner, 1990a, b, 1992). Such changes in O2-

demand of soybean bacteroids have been attributedto variations in the relationships betwen substrate uti-lization via the TCA cycle and deposition and utiliza-tion of PHB, the major C-reserve material which isprominent in soybean bacteroids (Bergersen and Turn-er, 1990b, 1992; Day et al., 1994). When C-substratesare in excess, PHB accumulates. When [O2] rises inresponse to diminished O2-demand because externalsources of substrate are in short supply (Bergersen,1991; Day et al., 1994), PHB is utilized in support ofrespiration and N2fixation. This property of bacteroidsmay be important for persistence of N2-fixation dur-ing pod-filling of soybeans (Bergersen et al., 1991);between strains of B.japonicum there is variability inPHB accumulation/utilization (Bergersen et al., 1994).

In flow chamber reactions using suspensions ofbacteroids isolated from soybean root nodules (Berg-ersen and Turner, 1992) and sesbania root and stemnodules (Bergersen et al., 1986), steady state ratesof N2 fixation are linearly correlated with respiratoryO2 consumpion rates. However certain limits apply.(i)There is a basal level of respiration below whichthere is little or no N2 fixation. This may correspondto part of the nodule’s "maintenance respiration" (Wit-ty et al., 1983). (ii)When a substrate becomes limit-ing, the relationship becomes non-linear. (iii) As [O2]approaches saturation of the terminal oxidase, N2 fixa-tion often declines linearly with further increases in O2

consumption (Bergersen, 1994, 1997). The relation-ships between bacteroid respiration and N2 fixationwere used to construct profiles of N2 fixation withininfected cells and to examine the effects of changesin substrate supply, [O2] at the cell surface and otherparameters in the model cell (Bergersen, 1994). Bac-teroid N2 fixation was able to proceed at high ratesover an unexpectedly wide range of cell surface [O2](Bergersen, 1994, 1996).

It is well-established that nitrogenase is rapidlyinactivated by excess O2 (e.g. Robson and Postgate,1980) and the inhibitory effects of relatively highO2 concentrations are often attributed to irreversibledestruction of nitrogenase (e.g. King and Layzell,1991). However, moderate periods of exposures ofbacteroids to high levels of O2 may not inactivatenitrogenase but rather initiate down-regulation of itsactivity; such regulation is often rapidly reversible(e.g. Bergersen and Turner, 1993). Figure 3 showsdata from a flow chamber experiment, in which anincrease in medium flow greater than could be abosrbedby increased respiration of soybean bacteroids (Figure3a), triggered a 60-fold increase in [O2], which was

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Figure 3. Data from a flow chamber (Bergersen, 1990) experi-ment (previously unpublished) with bacteroids (B.japonicum strainCB1809) prepared from soybean nodules. The bacteroids (53 mgd.wt.) in the chamber were perfused (at flows of 0.6, 1.0 and 0.9ml/min) with a reaction solution containing oxyleghaemoglobin(87 �M), dissolved air (230 �M O2), K-phosphate (2.5 mM)and mgSO4 (2 mM) in MOPS-KOH buffer, pH 7.4. At 212 min(arrow A), ethanol (22�M) an oxidisable substrate, was added tothe chamber and the reaction solution monitored spectrophotomet-rically for Lb oxygenation and the dissolved O2 concentration [O2]and rates of O2 consumption by the bacteroids were calculated asdescribed previously (Bergersen and Turner, 1990). Fixed N as NH+4in the effluent solution was assayed in fractions (5 min) of effluentsolution and the rates of N2 fixation calculated. (a) Time courses offlow rates (cont.line) and rates of bacteroid respiration (N). (b) Timecourses of changes in [O2] (line) and rates of N2 fixation (bars).

accompanied by an immediate decline in N2 fixation(Figure 2b). However, following a small decrease inflow rate, (Figure 3a), bacteroid respiration lowered[O2] rapidly and when a respirable C-substrate wasadded (at A, Figure 3a), bacteroid respiration increasedsharply and the consequent fall in [O2] below 50 nMwas followed immediately by restoration of N2 fixa-tion (Figure 3b). It is proposed that, as in aerobic dia-zotrophic bacteria, the regulated form of nitrogenaseis less-sensitive to irrevsrsible inactivation by excessO2. Mechanisms for such regulation have not yet been

Figure 4. Data from a flow chamber (Bergersen and Turner, 1990)experiment (previously unpublished) with bacteroids (B.japonicumstrain CB1809) from soybean nodules. The bacteroids (59.3 mg d.wt)in the chamber were perfused with a reaction solution containingdissolved air (235 �M O2) and oxyleghaemoglobin (107 �M) butdevoid of respirable substrate, otherwise as in the legend to Figure 2.At 268 min, after a steady state of> 30 min, the flow of medium wasreduced from 1.22 to 1.01 ml min�1. Measurements were made asdescribed in the legend to Figure 2. (a) Oscillations in respiration (N)induced by the flow rate change generated a controlled oscillation in[O2] (H), which was consequently maintained between limits of 13and 23 nM O2. (b) Oscillations in respiration were reflected in ratesof N2 fixation.

defined in this system but such regulation in other dia-zotrophic bacteria has been reviewed by Hill (1988).

Oscillations in respiratory activities have beenmentioned previously in this essay and in some caseshave been ascribed to cyclical changes in utilization ofendogenous PHB as a respiratory substrate (Bergersenand Turner, 1992). If bacteroid respiration is beingregulated during an apparent steady state in order tomaintain O2 homeostasis, control theory demands thatat least one reaction is being regulated between certainrate limits by some means which increases respirationwhen [O2] exceeds an upper limit and decreases respi-ration when [O2] falls too low. Usually, such oscilla-

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tions due to control limits are imperceptible during asteady state. However, a sudden perturbation may dis-turb the control mechanism sufficiently to change thetime constant of regulation and thus reveal oscillationsin rates. Such an effect is illustrated in Figure 4, againfrom a flow chamber experiment with soybean bac-teroids. After a period of >30 min of steady mediumflow, respiration and N2 fixation supported by utiliza-tion of endogenous PHB, a reduction in medium flowtriggered oscillations in bacteroid respiration whichproduced oscillations in [O2]. These were reflected inoscillations in N2 fixation which were slightly out ofphase. Often, such induced oscillations in respirationare damped progressively and soon become impercep-tible. However, in the experiment of Figure 4, theoscillations persisted beyond the time illustrated.

No account of the biology of N2 fixation in nod-ule cells would be complete without considering theeffects of H2 produced by nitrogenase and the impactof uptake hydrogenase. Since both activities are locat-ed in bacteroids, they are mentioned here. MolecularH2 is an unavoidable product of nitrogenase actionand is an inhibitor of N2 fixation, affecting the allo-cation of electrons between the substrates H+ and N2

(reviews Arp, 1992; Bergersen, 1991). It is now gen-erally agreed that, without uptake hydrogenase, H2

in nodule cells could reach inhibitory concentrations(e.g. Bergersen, 1991; Dixon, 1972; Hunt et al., 1988,Layzell et al., 1988, Moloney and Layzell, 1993). Oxi-dation of H2 by bacteroid uptake hydrogenase may addto the O2-demand generated by respiration, or spare C-substrate and by removing H2 from the nodule, willimprove the electron allocation to NH3 production bynitrogenase.

Conclusions

The above survey of literature, although far from beingexhaustive, is comprehensive enough to indicate that,in addition to the physical/structural barriers whichrestrict access of O2 from the atmosphere into legumeroot nodules, there are many biochemical and physio-logical components which may contribute to O2 home-ostasis. Some of these may prove to be componentsof the so-called ’variable diffusion barrier’ (Sheehyet al., 1983). Further, declines in nitrogenase activityinduced by various treatments which might be expect-ed to lead to inactivation of nitrogenase by excess O2,in part may be due to negative regulation of nitrogenaseactivity; such regulation is usually rapidly reversible

and should be considered when assessing the effects ofvarious stresses related to availability of O2.

Acknowledgements

The provision of a Fellowship by the AustralianNational University and the laboratory hospitality ofProfessor DA Day are gratefully acknowledged. Pro-fessor CA Atkins, University of Western Australia,generously made available unpublished material fromhis laboratory. The micrograph of Figure 1 was kindlysupplied by Celia Miller, CSIRO Microscopy Labora-tory, Canberra, using nodules embedded in 1972 andsectioned in 1993 for the study reported by Millar etal. (1995).

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Section editor: H Lambers

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