Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules

Annals of Botany 79 : 493–503, 1997

Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules E. K. J A M ES*, F. R. M I N C H I N†, P. P. M. I A N N E T TA‡ and J. I. S P R E N T Department of Biological Sciences, UniŠersity of Dundee, Dundee DD1 4HN, UK and † Institute of Grassland and EnŠironmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK Received : 26 September 1996

Accepted : 8 November 1996

The development of the N -fixing symbiosis between white lupin (Lupinus albus L.) cv. Multolupa and Bradyrhizobium # strain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy}immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. At 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA− (fix−) nodules at 17 d were smaller (mean radius of 0±49 mm compared to 1±01 mm with fix+ nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to be involved in both rhizobial infection and the regulation of nodule oxygen diffusion) gave a strong signal with nodules (fix+) from 20 d plants and with 17 d fix+ plants. The signal with MAC236}MAC265 was substantially weaker with nodules from 17 d fix− plants, and there was no signal apparent from nodules}nodulated roots from the 0, 8 and 12 d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix+}fix− nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC236 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N fixation. Nodules at all harvests were investigated for the presence of # infection threads, as the MAC236}MAC265-recognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the MAC236}MAC265 detected in lupin nodule extracts originated from glycoprotein within cortical intercellular spaces. # 1997 Annals of Botany Company Keywords : Lupinus albus, Bradyrhizobium, nitrogen fixation, nitrogenase, oxygen diffusion, glycoprotein, infection threads.

INTRODUCTION This paper is part of a series of studies examining the structure and regulation of oxygen diffusion into white lupin (Lupinus albus L.) nodules (Minchin et al., 1992 ; de Lorenzo et al., 1993 ; Iannetta et al., 1993 a, 1995 ; Witty and Minchin, 1994). The fine regulation of the influx of oxygen into nodules has a key role in the expression and functioning of the enzyme, nitrogenase, in legume nodules (Witty et al., 1986 ; Hunt and Layzell, 1993 ; Batut and Boistard, 1994 ; Soupene et al., 1995) as the enzyme is inactivated by oxygen concentrations of 5 µ and above (Sheehy and Thornley, 1988). However, there is also a requirement by the nitrogenase-containing bacteroids for aerobic respiration to * Current address for correspondence : Plant Sciences Laboratory, Sir Harold Mitchell Building, University of St. Andrews, St. Andrews, Fife KY16 9AL, UK. ‡ Present address : Mylnefield Research Services Ltd., Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.

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supply the high energy needs of N fixation (Witty et al., # 1986 ; Sheehy and Thornley, 1988 ; Gallon, 1992 ; Hunt and Layzell, 1993). Therefore, nodules have evolved various mechanisms to resolve the apparently conflicting requirements of aerobic respiration and protection of nitrogenase from O -mediated damage. One of these mechanisms is a # variable oxygen diffusion barrier in the nodule cortex that regulates the influx of external O into the N -fixing central # # part of the nodule so that the pO in the latter is maintained # at 10–20 n, i.e. giving sufficient dissolved O to supply (via # the O -carrying protein, leghaemoglobin) aerobic bacteroid # respiration without damaging nitrogenase (Hunt and Layzell, 1993 ; Witty and Minchin, 1994). Recent evidence suggests that in white lupin nodules the oxygen flux to the infected cells is across a water-filled diffusion barrier made up of cortical cells and variablyoccluded intercellular spaces, with no continuous air-filled pathways traversing the barrier (Witty and Minchin, 1994). Lupin nodules, and possibly soybean nodules (James et al.,

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1991 ; Iannetta et al., 1993 b), alter the influx of oxygen to the infected cells by varying the extent of intercellular space occlusions, the latter being partly composed of material containing epitopes recognized by the monoclonal antibodies MAC236 and MAC265 (de Lorenzo et al., 1993 ; Iannetta et al., 1993 a, 1995). MAC236 and MAC265 recognize epitopes on host-derived glycoproteins involved in the initial infection of legumes, and are expressed in infection threads and infection droplets (Bradley et al., 1988 ; VandenBosch et al., 1989 ; James et al., 1992, 1996 ; Rae, Bonfante-Fasolo and Brewin, 1992 ; Perotto, Brewin and Kannenberg, 1994), as well as in intercellular spaces in the cortex of mature nodules (Bradley et al., 1988 ; VandenBosch et al., 1989 ; James et al., 1991, 1992, 1993, 1994, 1996 ; Rae et al., 1991). Previous work in our laboratories has demonstrated a connection between cortically-localized MAC236 and MAC265 antigens and the regulation of oxygen influx into white lupin nodules during long-term (2 h–4 d) treatments, which produced measurable alterations in the oxygen diffusion barrier (Minchin et al., 1992 ; de Lorenzo et al., 1993 ; Iannetta et al., 1993 a). These treatments resulted in increases in extractable glycoprotein, as well as increases in the occlusion of intercellular spaces in the cortex, the postulated site of the oxygen diffusion barrier (Tjepkema and Yocum, 1974 ; Witty, Skøt and Revsbech, 1987 ; Parsons and Day, 1990 ; Dakora and Atkins, 1991 ; Serraj et al., 1995). Moreover, recent data from Iannetta et al. (1995) have shown that lupin nodules will occlude rapidly their cortical intercellular spaces by glycoprotein in response to a 15 min exposure to 50 % O , strongly # suggesting that glycoproteins have a role in lupin nodule O # regulation over the short-term as well as the long-term. However, the above studies concentrated on mature, N # fixing nodules in which nitrogenase activity was already expressed, and the oxygen diffusion barrier was also operational. It is now well established that there is a need for O regulation to create a microaerobic environment # during the early stages of nodule production to allow expression of nif and fix genes (Sheehy and Thornley, 1988 ; Soupene et al., 1995). Thus, if intercellular occlusions are an essential feature of O diffusion control in lupin nodules, the # matrix glycoproteins (MAC 236}MAC 265 antigens) should be expressed in developing nodules prior to the onset of nitrogenase activity. The main aim of the present study was to examine this point by determining the temporal relationships between the production of intercellular space glycoprotein in the nodule cortex and the expression of nitrogenase by the bacteroids. A further aim of this study was to examine L. albus nodules at various developmental stages to determine the presence of infection threads within them. Surrounding the bacteria within infection threads in many nodules, e.g. those on pea (Pisum satiŠum), soybean (Glycine max), Phaseolus Šulgaris, Vicia faba, Neptunia and Sesbania, there is a matrix which is also recognized by MAC236 and MAC265 (Bradley et al., 1988 ; VandenBosch et al., 1989 ; James et al., 1992, 1996 ; Rae et al., 1992). The extent of MAC236}MAC265 within infection threads in lupin nodules needs to be established as crude extracts of L. albus nodules, subjected to various treatments, have been used in comparative Western

blot and ELISA analyses of MAC236}MAC265 (de Lorenzo et al., 1993 ; Iannetta et al., 1993 a, 1995). In these analyses it has been assumed that the MAC236}MAC265 is entirely derived from cortical glycoprotein rather than from the matrices of infection threads, as the latter are apparently scarce in mature lupin nodules (Jordan and Grinyer, 1965 ; Dart, 1977 ; Tang et al., 1992, 1993). MATERIALS AND METHODS Plants of white lupin (Lupinus albus) cv. Multolupa were grown in a glasshouse as described by James et al. (1993). Seeds were germinated in pots of vermiculite, inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16 and watered with nitrogen-free nutrient solution (James et al., 1991). Roots were analysed 8, 12, 17 and 20 d after inoculation for the presence or absence of nitrogenase activity using the ‘ closed ’ acetylene reduction assay (James et al., 1991). In addition, roots with macroscopically visible nodules and nodule primordia were fixed and sectioned for microscopy using the procedures of James et al. (1991), i.e. fixed in 2±5 % glutaraldehyde, dehydrated in an ethanol series and embedded in LR White resin (London Resin Co.). Nodules from plants that did not express any ARA and which had no visible pink coloration (due to leghaemoglobin, Lb) were considered to be at the pre-N # fixation stage of development (Bisseling et al., 1980), whereas nodules from plants with significant ARA and with visible Lb were considered to be expressing nitrogenase. This was confirmed in individual nodules using immunogold labelling for light and transmission electron microscopy (TEM) with a polyclonal antibody against nitrogenase component II (from Rhodospirillum rubrum) according to James et al. (1996). Immunogold labelling with the monoclonal antibodies MAC236 and MAC265 was performed according to VandenBosch et al. (1989) and James et al. (1991). The control for the monoclonal antibody immunogold labelling was omission of MAC236 or MAC265, and the controls for the polyclonal nitrogenase antibody were omission of the latter or substitution of normal rabbit serum (Sigma) for the nitrogenase antibody (see James et al., 1996). Protein in nodules and roots at each harvest (six plants per harvest) were immediately extracted (0±1 g fresh weight of material to 1 ml buffer) using the buffer solution described by Bradley et al. (1988) and VandenBosch et al. (1989) and used in a previous study of MAC236}MAC265 in lupin nodules (de Lorenzo et al., 1993). At the 17 and 20 d harvests there was sufficient nodule mass for the nodules to be separated from the roots, but this was not the case at the 0, 8 and 12 d harvests where the nodules were very small and were embedded within the subtending roots (Figs 2 A, 3 A). Therefore, pieces of nodulated roots were extracted instead. Protein in the samples was measured according to James et al. (1991), separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (12 % gels), and Western blotted onto nitrocellulose according to de Lorenzo et al. (1993). MAC236 and MAC265 antigens on the Western blots were detected using 1}100 dilutions of the antibodies according to VandenBosch et al. (1989) and James et al. (1996).

James et al.—Temporal Relationships between Nitrogenase and Intercellular Glycoprotein RESULTS Nitrogenase actiŠity and determination of MAC236} MAC265 in nodule extracts Nitrogenase activity (ARA) giving ethylene values above that of background (0±05 µmol C H per plant h−") was not # % detected in any of the lupin plants until the 17 and 20 d harvests. However, at the 17 d harvest only three of the six plants tested had ARA significantly above that of background (mean : 0±45³0±01 µmol C H per plant h−"), and # % this rose to four out of six plants at the 20 d harvest (mean : 0±46³0±33 µmol C H per plant h−"). Nodulated roots from # % the 0, 8 (not shown) or 12 d harvests (Fig. 1) did not contain sufficient concentrations of the MAC236 or MAC265 epitopes for them to be detected by Western analysis. However, both glycoprotein epitopes were detectable in nodule extracts from the 17 and 20 d harvests, the concentrations of both epitopes (and of soluble protein) being greatly enhanced in fix+ compared to fix− nodules at the 17 d harvest (Fig. 1). The molecular weight of MAC236 was approx. 240 kDa in both fix+ and fix−nodule extracts (Fig. 1 A). The molecular weight of the MAC265 antigen was between 95 and 135 kDa in the fix+ nodule extracts, although only a band at 135 kDa was visible in the fix− extracts (Fig. 1 B). These weights are in general agreement with those reported previously for this symbiosis by de Lorenzo et al. (1993). Immunogold localization of MAC236, MAC265 and nitrogenase component II By 8 d after inoculation nodule primordia were initiated in cells immediately beneath the root epidermis, and these primordia contained numerous, newly-infected, meristematic cells (Fig. 2 A). MAC236 was strongly localized in material occluding intercellular spaces in uninfected tissue surrounding the nodule primordia, particularly in the tissue destined to develop into the nodule cortex (Fig. 2 B and C). MAC265 was also localized within material in these spaces (Fig. 2 D). At 12 d after inoculation, nodules were multilobed and the central tissue was composed of rapidly dividing A

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infected cells (Fig. 3 A and B), with numerous MAC236labelled occlusions between both the cortical (Fig. 3 C) and infected cells (Fig. 3 D). The infected tissue in 8 and 12 d nodules consisted entirely of closely-packed, highly vacuolate cells, with no uninfected cells apparent, and neither the meristematic nodule primordia at 8 d (Fig. 2 A and B) or the young nodules at 12 d (Fig. 3 A, B and C), had any obvious infection threads. The bacteria in nodules at 8 and 12 d (e.g. those in Fig. 3 D) did not label with the antibody to nitrogenase component II. The sections in Fig. 4 A and B are representative of 17 d fix− and fix+ nodules, respectively. The mean radius of the five largest fix− nodules examined at this harvest was 0±49 mm (³0±05 s.e.), and that of the five smallest fix+ nodules was 1±01 mm (³0±07). Bacteroids within the fix− nodules did not express nitrogenase component II (Fig. 4 C and D) whereas those in fix+ nodules did (Fig. 4 E and F). At 17 d the cortices of both fix− and fix+ nodules had intercellular spaces containing the glycoprotein recognized by MAC236 (arrows, Fig. 5 A and B). In fix+ nodules the different cortical zones previously characterized for this symbiosis by Fernandez-Pascual et al. (1992), de Lorenzo et al. 1993 and Iannetta et al. (1993 a, 1995) were clearly evident (Fig. 5 A and B). These were : zone 1, the outer cortex ; zone 2, 2–3 layers of cells in the mid-cortex with thick walls and most of the intercellular glycoprotein occlusions (Fig. 5 C and D) ; zone 3, the inner cortex adjacent to the infected zone, comprising 2–3 layers of cells in a combined boundary layer}distribution zone (Witty et al., 1987 ; Parsons and Day, 1990). Also apparent in fix+ nodules was thickening of the cell walls in zone 2 (Fig. 5 B and D). However, the more meristematic nature of the cortex in fix− nodules (Fig. 5 A) did not allow for quantification of this feature. The cortices of 20 d fix+ nodules were similar in structure to those of 17 d fix+ nodules (Fig. 5 B), and they also expressed glycoproteins within their cortical intercellular spaces (not shown). The meristems of 17 and 20 d nodules did not have any obvious infection threads (not shown), and resembled the meristems in 8 and 12 d nodules (Figs 2 A and B ; 3 A and B). However, serial sectioning of 20 d nodules occasionally B

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84 41 F. 1. Western blots of protein extracts from equal fresh weights of lupin nodules}nodulated roots at various harvests (12, 17 and 20 d after inoculation). Each lane contains 5 µl of extract and the concentration of soluble protein in the extracts (mg g−" fresh weight) was 2±3 (12 d fix−), 7±6 (17 d fix−), 11±7 (17 d fix+) and 9±5 (20 d fix+). The extracts were designated fix+ if they came from plants with significant acetylene reduction activity (ARA), or fix− if the plants had no ARA. Blots were probed with the monoclonal antibody MAC236 (A) or MAC265 (B). The positions of the molecular weight markers (BioRad) are indicated on the left (Mr¬10−$ kDa).

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F. 2. A, Nodule primordium emerging from the epidermis of a root of Lupinus albus 8 d after inoculation with Bradyrhizobium strain ISLU16. At the tip of the primordium is a small crack in the epidermis (asterisk). Newly infected meristem cells (M, grey cells) have formed immediately beneath the epidermal crack. A root hair is indicated by an arrow. Bar ¯ 50 µm. B, Nodule primordium at 8 d after inoculation ; the radius of the nodule is 0±16 mm. This section was immunogold-labelled with the monoclonal antibody MAC236 followed by silver enhancement. The glycoprotein recognised by MAC236 can be seen accumulated at the tip of the nodule (asterisk), and also within intercellular spaces (arrows) in

James et al.—Temporal Relationships between Nitrogenase and Intercellular Glycoprotein revealed small, isolated pockets and files of uninfected cells within the infected region (Fig. 6 A) and bacterial invasion was apparently occurring within these (Fig. 6 B). Under the TEM it was shown that bacteria were indeed invading the host cells in these nodules via short infection threads (Fig. 6 C and D). These infection threads had a matrix which either labelled very sparsely (Fig. 6 D), or not at all (not shown), with MAC236 and MAC265. This type of cell invasion was not seen in serial-sectioned nodules from any of the earlier harvests. However, infection thread-like structures within nodule primordia at 8 d after infection were occasionally observed, although the exact nature of these is still to be confirmed by electron microscopy. Control sections for immunogold labelling with MAC236, MAC265 and nitrogenase component II showed no labelling (not shown). DISCUSSION Expression of nitrogenase and cortical glycoprotein Sheehy and Thornley (1988) and Soupene et al. (1995) have demonstrated that rhizobia in nodules will only synthesize nitrogenase when the pO of the developing nodule has # decreased to a level at which nifA can be expressed ; this involves both the formation of an oxygen diffusion barrier and O consumption by bacteroid respiration to maintain a # pO below 1 µ (Witty et al., 1986 ; Hunt and Layzell, # 1993). In addition, Bisseling et al. (1980) showed that the synthesis of the O -carrying protein, Lb also precedes the # expression of nitrogenase, further suggesting that a low pO # is an essential prerequisite. In the present study of L. albus, nitrogenase was not expressed until some time between 12 and 17 d after inoculation, suggesting that the aforementioned criteria were established, at least in some nodules, during this period. Hence our study agrees with that of Halvorson et al. (1991), who reported nitrogenase activity by perennial Lupinus spp. 14 d after germination, and with that of Trinick, Dilworth and Grounds (1976), who did not observe N fixation in lupin nodules until after 17 d. # Interestingly, the work of Bisseling et al. (1980) and Vasse et al. (1990) shows that the time scale for nitrogenase component II expression in Lupinus nodules may be slightly later than that of other symbioses. For instance, Vasse et al. (1990) reported pea nodules with fully-differentiated bacteroids expressing nitrogenase activity at 8 d after inoculation, and Bisseling et al. (1980) showed that nitrogenase component I was expressed in the bacteroids from their pea-Rhizobium symbiosis at 10 d after inoculation, and that component II was expressed 1–2 d later. However, nitrogenase in both lupin and pea nodules is expressed considerably later than that in Sesbania rostrata nodules, e.g. Ndoye et al. (1994) have recently reported (symbiotic) acetylene reduction by 3-d-old root nodules on this species.

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Sheehy and Thornley (1988) calculated that 0±91 mm is the theoretical minimum radius at which the microaerobic conditions necessary for the induction of the nifA gene can be achieved. In the present study, the fix− nodule primordia at 8 d (Fig. 2 A and B) and 12 d (Fig. 3 A and C) had radii considerably smaller than this (0±18 and 0±30 mm, respectively). The bacteria within the smaller nodules on 17 d plants (e.g. Fig. 4 A and C ; radius of 0±60 mm) also did not express nitrogenase, whereas the larger ones did (e.g. Fig. 4 B and D ; radius of 1±25 mm). Therefore, L. albus nodules appear to fit the model of Sheehy and Thornley (1988) in requiring a radius somewhere between 0±6 and 1±2 mm before nitrogenase is expressed. Indeed, in an earlier study, Trinick et al. (1976) also observed that nitrogenase activity did not occur in L. angustifolius and L. cosentinii nodules until they exceeded a diameter of 1±5 mm. Interestingly, this may not be the case with nodules from other symbioses, as nitrogenase activity has been demonstrated in soybean and pea nodules with radii of less than 0±4 mm (Bergersen and Goodchild, 1973 ; Vasse et al., 1990). A simple increase in nodule radius will not be the only factor involved in the expression of nitrogenase within nodules as there must also be a very high rate of respiratory O consumption within the developing nodule, and this is # likely to be supplied mainly by high meristematic respiration until the induction of nitrogenase-linked respiration (Sheehy and Thornley, 1988). Moreover, the establishment and maintenance of an O diffusion resistance in the cortex # surrounding the developing and expanding infected zone will also be an essential requirement if the central zone pO # is to be reduced via respiratory O consumption. The most # obvious way to achieve this diffusion resistance is to occlude the air-filled pathways through the cortex so that O has to # diffuse through water-filled pathways, e.g. across cells (Dakora and Atkins, 1991 ; Witty and Minchin, 1994 ; Serraj et al., 1995). There is much recent evidence from mature, actively fixing nodules of the L. albus}ISLU16 symbiosis that cortical air spaces in this symbiosis are occluded with the glycoprotein recognized by MAC236}MAC265 (de Lorenzo et al., 1993 ; Iannetta et al., 1993 a, 1995) ; and the present study suggests this is also the case in developing, pre-N -fixing L. albus}ISLU16 nodules. # However, there are apparently conflicting results with MAC236}MAC265 using Western blot analyses of total nodule}nodulated root extracts on the one hand, and immunogold labelling on the other. Western blots showed that there was apparently no MAC236}MAC265 in 0, 8 and 12 d nodules, whereas microscopy showed that MAC236 and MAC265 were actually expressed quite strongly in nodule primordia from the 8 and 12 d harvests. This difference is probably due to the lack of sensitivity of Western blot analysis compared to immunogold labelling. The latter, by its very nature, is a micro-technique and inherently more sensitive and ideal for the qualitative

the uninfected (cortical) tissue surrounding the newly-infected nodule meristem (M). Bar ¯ 50 µm. C, Transmission electron micrograph (TEM) of an intercellular space from the developing cortex surrounding a nodule primordium at 8 d after inoculation (see Fig. 2 B). The space is occluded with material immunogold labelled with MAC236 (asterisk). Bar ¯ 200 nm. D, Cell wall-associated material (immunogold labelled with MAC265 ; arrows) within a large intercellular space in the developing cortex at the base of a nodule primordium at 8 d after inoculation. C, Host cell wall. Bar ¯ 200 nm.

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F. 3. A, Lupin root (R) with a nodule primordium at 12 d after inoculation. This section shows 3 lobes of infected cells (grey cells) developing in one nodule, and the largest of them (asterisk) has a radius (including the surrounding cortex) of 0±3 mm. Bar ¯ 100 µm. B, Detail of the meristematic cells in a developing nodule primordium at 12 d after inoculation. Many of the cells are actively dividing, some with visible chromosomes (arrows), and all of the cells in the infected region are infected with rhizobia. No infection threads were observed in nodules at this stage. Bar ¯ 10 µm. C, Lobe of a 12 d nodule (see Fig. 3 A) immunogold labelled with MAC236 followed by silver enhancement. Most of the MAC236 antigen is localized within intercellular spaces (arrows) in the cortex surrounding the lobe of infected cells (asterisk). Bar ¯ 50 µm. D, Intercellular space (asterisk) within the infected zone of a nodule at 12 d after inoculation ; the space is occluded with material recognized by MAC236. In adjacent serial sections the bacteroids (B) did not label with an antibody raised against nitrogenase component II. S, Hole left by a starch grain. Bar ¯ 500 nm.

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F. 4. A, Longitudinal section (LS) of a root plus nodule at 17 d after inoculation. This nodule has a radius of 0±6 mm and was taken from a plant which had no nitrogenase activity (ARA). Bar ¯ 100 µm. B, LS of a nodule at 17 d after inoculation, taken from a plant with significant ARA. This nodule has a radius of 1±25 mm. Bar ¯ 200 µm. C, Section from the arrowed region of the 17 d fix− nodule shown in Fig. 4 A. This section was immunogold labelled with an antibody against nitrogenase component II and silver enhanced. There is no labelling of the infected cells, the signal (solely derived from a light background staining with toluidine blue) is similar to a negative control with normal rabbit serum substituted for the nitrogenase antibody (not shown). Bar ¯ 50 µm. D, TEM of bacteroids from a 17 d fix− nodule. This section was immunogold labelled with an antibody against nitrogenase component II but there is no gold labelling of the bacteroids (arrows). Bar ¯ 500 nm. E, Section from the arrowed region of the 17 d fix+ nodule shown in Fig. 4 B. This section was immunogold labelled with an antibody against nitrogenase component II and silver enhanced. The bacteria within the infected cells are strongly labelled with the antibody. Bar ¯ 50 µm. F, TEM of bacteroids from a 17 d fix+ nodule. This section was immunogold labelled with an antibody against nitrogenase component II and the bacteroids are labelled (arrows). Bar ¯ 500 nm.

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F. 5. A, Transverse section from the cortex of a 17 d fix− nodule. This section was immunogold labelled with MAC236, followed by silver enhancement. The glycoprotein is primarily localized in the intercellular spaces (arrows) within the mid-cortex region (MC). IT, Infected tissue. Bar ¯ 10 µm. B, Transverse section from the cortex of a 17 d fix+ nodule showing part of the outer cortex (zone 1), the thick-walled cells of zone 2, the boundary layer}distribution zone cells of zone 3 and the outer edge of the infected tissue (IT). This section was immunogold labelled with MAC236, followed by silver enhancement and the glycoprotein is primarily localized in the intercellular spaces (arrows) within cortical zone 2 (analogous to the mid-cortex in Fig. 5 A). Bar ¯ 10 µm. C, Intercellular space from the mid-cortex of a 17 d fix− nodule. The space is partially occluded with material immunogold-labelled with MAC236. C, Cell wall. Bar ¯ 500 nm. D, Intercellular space from zone 2 of a 17 d fix+ nodule. The space is occluded with material immunogold-labelled with MAC236. C, Cell wall. Bar ¯ 200 nm.

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F. 6. A, Section of a fix+ nodule at 20 d after inoculation. This shows large ‘ pockets ’ (white arrows) of uninfected cells within the infected zone. The meristem (M) is visible on the periphery of the infected zone. Bar ¯ 100 µm. B, Section of a pocket of uninfected cells in a nodule at 20 d after inoculation. There are large groups of intercellular bacteria (arrows) associated with the uninfected cells. An infection thread-like structure appears to be entering one of these cells (large arrow). Bar ¯ 10 µm. C, TEM of a pocket of uninfected cells within the infected tissue of a nodule at 20 d after inoculation. The cells are being invaded by infection threads (arrows), which appear to originated from intercellular spaces which contain bacteria (large arrows). Bar ¯ 5 µm. D, Detail of an infection thread and infection droplets from Fig. 6 C. Bacteria are being released into the cell at thin-walled regions of the infection thread (arrows). The matrix of the infection thread is very sparsely immunogold labelled with the monoclonal antibody MAC265 (small arrows). Bar ¯ 500 nm.

analysis of very small samples such as the nodule primordia used in the present study. Moreover, the 0, 8 and 12 d extracts had a high ratio of root : nodule tissue as nodulated roots had to be used rather than nodules, and the very low

concentrations of the epitopes in roots (Bradley et al., 1988 ; VandenBosch et al., 1989 ; Rae et al., 1991) would have diluted the relatively higher concentrations of MAC236} MAC265 in the nodule primordia.

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James et al.—Temporal Relationships between Nitrogenase and Intercellular Glycoprotein

Nevertheless, in the present study both these techniques have demonstrated unambiguously that expression of MAC236 and MAC265 within intercellular occlusions in the developing cortices of young lupin nodules occurs before the onset of nitrogenase expression and activity. The 8 and 12 d nodule primordia showed very strong localization of MAC236}MAC265 within the intercellular spaces immediately surrounding the newly-infected tissue (Figs 2 B, 3 C), i.e. the region where the diffusion barrier would need to develop. Further evidence for the involvement of glycoprotein in the operation of a cortical O diffusion barrier # in young lupin nodules is provided by the 17 d harvest. At − + this time, both the fix and fix nodules had most of their glycoprotein localized within the cortical region previously identified as the main location of the oxygen diffusion barrier in this symbiosis, i.e. zone 2 or the mid-cortex (de Lorenzo et al., 1993 ; Iannetta et al., 1993 a, 1995). Then again, qualitative analyses of the Western blots showed that extracts of actively N -fixing nodules contained considerably # greater concentrations (on a fresh weight basis) of the glycoprotein compared to nodules not yet expressing nitrogenase, and that this increase coincided with an increase in nodule soluble protein (Fig. 1 A and B). Taken together, these results demonstrate that production of the intercellular glycoprotein recognized by MAC236} MAC265 precedes the expression of nitrogenase activity in young L. albus nodules. This is a prerequisite for any component of the nodular O diffusion barrier and provides # further support to the hypothesis that the MAC236} MAC265 antigens are a major feature of this barrier. The role of other intercellular components, such as diprenylated isoflavones (Grandmaison and Ibrahim, 1995), lectins (VandenBosch et al., 1994) and proline-rich proteins (Sherrier and VandenBosch, 1994) in the establishment of the diffusion barrier in lupins and other legume nodules now needs to be investigated. Infection thread glycoprotein Another point brought out by this study is that infection threads are very scarce in both developing (8, 12 and 17 d) and N -fixing (17 and 20 d) lupin nodules, and were only # observed in the present study after serial sectioning of nodules. The scarcity of infection threads in lupin nodules is also reinforced by the unpublished data of James and Iannetta who sectioned over 50 mature L. albus nodules during the study of Iannetta et al. (1995) and found obvious infection threads in only three of them. Moreover, the infection threads were only found in younger nodules, and in all cases these were within the infected tissue behind the meristem, rather than within the meristem itself. Therefore, the present study is in general agreement with Jordan and Grinyer (1965), Dart (1977) and Tang et al. (1992, 1993) who have all reported that infection threads are very scarce in lupin nodules, particularly in older nodules. The infection threads and infection droplets observed here within L. albus nodules were very sparsely labelled with MAC236 and MAC265, unlike those reported in other indeterminate root nodules (VandenBosch et al., 1989 ; James et al., 1992 ; Rae et al., 1992 ; Perotto et al., 1994), or those

in S. rostrata stem and root nodules (James et al., 1996). This suggests that lupin nodule infection threads have a different matrix composition. Indeed, the present study and those of Robertson et al. (1978) and Tang et al. (1993) suggest that infection threads in Lupinus nodules more closely resemble those in determinate nodules, such as soybean and Phaseolus, i.e. relatively short structures originating from between cell walls, not traversing cells (Newcomb, Sippell and Peterson, 1979 ; Rae et al., 1992 ; James et al., 1993), and containing little or no matrix glycoprotein (VandenBosch et al., 1989 ; Rae et al., 1992). Therefore, in the present study and in the studies of de Lorenzo et al. (1993) and Iannetta et al. (1993 a, 1995), in which whole white lupin plant nodule populations were taken for ELISA and Western blot analysis of relative glycoprotein contents, nearly all of the glycoprotein measured will have been from cortical cells and}or intercellular spaces, as that within infection threads is so low. Extracts of indeterminate nodules such as pea (Pisum satiŠum L.), Vicia faba (L.) and lucerne (Medicago satiŠa L.) contain a considerable amount of glycoprotein from infection threads and infection droplets (VandenBosch et al., 1989 ; James et al., 1992 ; Rae et al., 1992, Perotto et al., 1994), perhaps accounting for over 50 % of the total glycoprotein measured in the nodules (Brewin, pers. comm.). Therefore, when attempting to measure changes in glycoprotein contents of whole nodule extracts from indeterminate nodules, such as those from lucerne (Hunt et al., 1995 ; Wycoff et al., 1995), care must be taken before conclusions about purely cortical glycoprotein can be made, as there will be a background pool of infection thread} infection droplet glycoprotein. A C K N O W L E D G E M E N TS We thank Dr N. J. Brewin, John Innes Centre, Norwich, UK for the MAC236 and MAC265 monoclonal antibodies and Dr P. W. Ludden, Madison, Wisconsin, USA for antibodies to nitrogenase. N. J. Brewin and J. M. Sutherland are thanked for helpful discussions and E. L. Fox, M. Gruber, M. Kierans, C. James and H. Hodge for their technical assistance. E. K. James was funded by NERC, and P. P. M. Iannetta by an AFRC co-operative studentship. LITERATURE CITED Batut J, Boistard P. 1994. Oxygen control in Rhizobium. Antonie Šan Leeuwenhoek 66 : 129–150. Bergersen FJ, Goodchild DJ. 1973. Cellular location and concentration of leghaemoglobin in soybean root nodules. Australian Journal of Biological Sciences 26 : 741–756. Bisseling T, Moen AA, Van den Bos RC, Van Kammen A. 1980. The sequence of appearance of leghaemoglobin and nitrogenase components I and II in root nodules of Pisum satiŠum. Journal of General Microbiology 118 : 377–381. Bradley DJ, Wood EA, Larkins AP, Galfre G, Butcher GW, Brewin NJ. 1988. Isolation of monoclonal antibodies reacting with peribacteroid membranes and other components of pea root nodules containing Rhizobium leguminosarum. Planta 173 : 149–160. Dakora FD, Atkins CA. 1991. Adaptation of nodulated soybean (Glycine max L. Merr.) to growth in rhizospheres containing nonambient pO . Plant Physiology 96 : 728–736. # Dart P. 1977. Infection and development of leguminous nodules. In :

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