Plant material and growth conditions
Seeds of soybean (Glycine max (L.) Merr. cv. Clarke) were inoculated at sowing, and then after 7 d, with Bradyrhizobium japonicum strain RCR 3407. Seeds of pea (Pisum sativum L.) cv Kelvedon Wonder were inoculated with Rhizobium leguminosarum bv. viciae leg29d. Plants were grown in a Saxcil growth cabinet providing 16 h light (approximately 600 μmol m–2 s–1) set at 25/15 °C and were watered daily with N-free nutrient solution. At 42 d after sowing, soybeans were subjected to a detopping (shoot removal) treatment. Nodules were then harvested from these plants as well as unstressed soybeans and peas.
Nodules were harvested from six replicate plants of soybean and pea 3 h after detopping or from six replicate unstressed (control) plants. A 200 mg fresh weight subsample from each plant was then frozen in liquid N2 and stored at − 80 °C until used for Western blotting, cryo-sectioning or ELISA. For soybeans, 10 nodules from each plant were fixed for microscopy.
Protein in nodules was extracted (0·1 g fresh weight to 1 mL buffer) using the buffer solution described by VandenBosch et al. (1989) and also used in a previous study of MAC236/MAC265 in lupin nodules ( de Lorenzo et al. 1993 ). 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 a 1/100 dilution of the antibodies according to James et al. (1996) .
Quantification of glycoprotein using enzyme-linked immunosorbent assay (ELISA)
This involved the extraction and processing methods of de Lorenzo et al. (1993) and the ELISA protocol of Iannetta et al. (1993a) but used the MAC265 antibody instead of MAC236 as it gave a stronger colour development in denatured extracts. Results are the means of six nodules per plant for six replicate plants; giving 36 estimates for each experimental treatment. All 36 estimates were analysed simultaneously to allow comparison of relative amounts of antigen. The ‘minimum dilution’ (MD) at which glycoprotein could be detected was recorded and the MD per g fresh weight of nodule tissue was determined for each assay. The ‘average minimum dilution’ (AMD g–1 fresh weight) for each treatment was then calculated from the 36 assays Statistical analyses were performed using ANOVA and Duncan’s multiple range tests.
Tissue printing and cryo-sectioning of unfixed nodules
Three frozen nodules from each treatment were sliced in half and immediately printed on to nitrocellulose (BioRad) according to Cassab & Varner (1987). The prints were incubated for 1 h in a 10% solution of skimmed milk in Tris-buffered saline (TBS) followed by a 2 h incubation in a 1/50 dilution of either MAC236 or MAC265. After washing in TBS + Tween-20, the prints were incubated for 1 h in a 1/1000 dilution of anti-rat alkaline phosphatase conjugate (Sigma), and the signal was visualized using a BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution (Sigma) as per Western blots ( James et al. 1996 ).
For cryo-sectioning, frozen nodules were embedded in Tissue Tek (Agar Aids, Stansted, UK) and sectioned at − 20 °C on a Reichert-Jung Cryocut E using a stainless steel knife. The sections (20 μm thick) were collected on glass slides and incubated at room temperature for 1 h in the blocking buffer used in immunogold labelling of resin-embedded material ( James et al. 1991 ). The sections were then incubated for 2 h in MAC236 or MAC265 (the antibodies being diluted in the blocking buffer at 1/100). After gentle washing in blocking buffer, the sections were incubated in anti-rat alkaline phosphatase as above.
Blocking buffer was substituted for MAC236/MAC265 as a negative control for the labelling of the tissue prints and the cryo-sections. In the case of the cryo-sections, both controls and immunolabelled sections were lightly background-stained with toluidine blue according to James et al. (1991) .
Microscopy and immunogold labelling
Immunolabelling of glycoproteins for light and transmission electron microscopy (TEM) was performed with MAC236 and MAC265 according to VandenBosch et al. (1989) and James et al. (1991) . Both antibodies have previously been shown to react with intercellular glycoproteins in a wide range of legume nodules ( James et al. 1994 ; Brown & Walsh 1996).
Morphometric analysis of the nodule cortex
Quantification of intercellular space occlusions was carried out using image analysis methods. Nodules from control and detopped plants were processed for microscopy as detailed above, and cross-cortex sections were toluidine blue-stained and photographed under an Olympus BH2 light microscope (× 20 magnification objective). The black and white micrographs were then trimmed to isolate the inner and mid-cortex region between the infected cells and the thick-walled scleroid cells (see Minchin 1997 for classification of nodule cortex components). The trimmed micrographs were scanned onto a white background and digitized in a tagged image file (TIF) format using a ScanJetII image scanner (Hewlett Packard, Amsterdam, The Netherlands) linked to DeskScanII software. Instrument scanning settings were: brightness and contrast, 125; scaling, 100%; and image ‘dark-tones enhanced’. The grey-scale TIF images were then processed using the ‘sharpen more’ facility of Paint Shop Pro (PSP) 3 software before being quantified using KS300 image analysis software (Imaging Associates Limited, Thames, UK).
Each digitized micrograph was segmented using the interactive threshold facility of the KS300 package. Thresholding set a value within a defined grey scale range of 0 (black) to 255 (white). Pixels with values greater than the threshold were re-assigned a value of 255, whilst pixels with values less than the threshold were re-assigned a value of 0. The image used for quantification was therefore a binary version of the input image.
Following the thresholding treatment, each TIF image consisted of a white background area plus the black and white area of the micrograph to be analysed. To allow quantification of the total section area, this was separated from the white background by a two-pixel wide black line. For the final stage, the white intracellular areas were coloured black using the ‘fill’ feature of PSP 3 to leave only the background and unoccluded intercellular spaces as white areas. These were measured individually using the image analysis software, and the background area, which was easily distinguishable as a very large value, was discarded. The remaining intercellular space area measurements were then counted to give the number of spaces per section and summed to give the total space area, which was expressed as a percentage of the total section area (white area minus background, plus black area minus the area of the perimeter line).
This procedure did not distinguish between cell walls and occluding material in the intercellular spaces since both were stained dark by toluidine blue and consequently were measured as part of the black area following thresholding. However, this occluding material may be immunogold-labelled with MAC236 and visualized for light microscopy by silver enhancement (SE-IgL, see above). Therefore, appropriate thresholding of silver-enhanced nodule sections would reveal the areas of MAC236 antigen which could then be quantified by image analysis. Sections of nodules from control and detopped plants were treated simultaneously using standardized conditions for SE-IgL with the MAC236 antibody followed by light staining with toluideine blue. Photographs of the sections were trimmed and scanned as described above, using scanning conditions of: scaling, 500%; brightness, 90; contrast, 180. The digital images were stored in TIF format and the files further processed using PSP 5 software to remove the lightly stained cell-wall material and only leave the black silver particles. For this, the colour depth of the image was increased to 16 million (24-bit), the ‘sharpen more’ option applied and the ‘colour replace’ tool then selected at a grey-scale value of 128. All grey shades lighter than 128 were converted to white and only dark pixels (silver-enhanced immunogold particles) remained. The images were finally ‘despeckled’, and, using image analysis, individual blocks of silver particles were measured as a single area values. At this stage, a threshold value was employed to exclude any single areas of four pixels or less. The localization of such excluded areas indicated (on comparison with the original micrograph) that they were dark staining cell-wall associated particulates. The sum of those areas greater than four pixels estimated the area of the section which was silver-enhanced; this was expressed as a percentage of the total section area.
For unoccluded intercellular space counts and areas, control nodules were quantified using 24 unlabelled micrographs, representing five nodules harvested from five different plants (five sections from each of four nodules and four sections from a 5th nodule). The effect of detopping was examined over 22 micrographs that represented six nodules harvested from six different plants (four nodules giving four sections each and two nodules each providing three sections). For silver-enhanced MAC236 areas, 12 micrographs were quantified from control and detopped nodule sections, representing two nodules each from three plants. Statistical analyses were carried out using one-way ANOVA.