*Present address: Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules
Article first published online: 25 DEC 2001
Plant, Cell & Environment
Volume 23, Issue 4, pages 377–386, April 2000
How to Cite
James, E. K., Iannetta, P. P. M., Deeks, L., Sprent, J. I. and Minchin, F. R. (2000), Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules. Plant, Cell & Environment, 23: 377–386. doi: 10.1046/j.1365-3040.2000.00549.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- oxygen diffusion;
A structural analysis was conducted to determine whether glycoprotein-containing intercellular space occlusions are involved in medium-term regulation of O2 diffusion in soybean (Glycine max) nodules. Alterations in O2 diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid-cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver-enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O2 diffusion regulation in soybean nodules
It is now generally accepted that legume nodules contain a restriction to the free diffusion of O2 from the atmosphere to the central, bacteroid-containing cells ( Tjepkema & Yocum 1974; Parsons & Day 1990), and that such a barrier is essential to prevent inactivation of the nitrogenase enzyme and/or excess production of activated oxygen species ( Sheehy & Thornley 1988; Escuredo et al. 1996 ). It is also widely accepted that the resistance of this oxygen barrier can be rapidly varied in response to external stimuli ( Witty et al. 1986 ; Hunt & Layzell 1993). This variability relies mainly upon physical changes within the nodule ( Witty & Minchin 1998), and probably includes the movement of water into intercellular spaces (ICS) within the nodule cortex. Studies with nodules under He/O2 and Ar/O2 environments suggest that, in unstressed soybean nodules, about half of the O2 flux to infected cells is via interconnected gas-filled pores, which ‘close’ to produce a liquid-filled barrier as the diffusion resistance increases in response to stress ( Witty & Minchin 1994). However, the nature and location of these physical changes within legume nodules is still a matter of controversy ( Hunt & Layzell 1993; Minchin 1997).
Studies on nodules of soybean ( James et al. 1991 ; Iannetta et al. 1993b ), lupin (Lupinus albus) ( de Lorenzo et al. 1993 ; Iannetta et al. 1993a ; Iannetta et al. 1995 ) and Lotus ( James & Crawford 1998; James & Sprent 1999) have suggested that one component of the diffusion barrier is the occlusion of cortical intercellular spaces with glycoproteins, recognized by the monoclonal antibodies MAC236 and MAC265, as first reported by VandenBosch et al. (1989) and Rae et al. (1991) . To date, the published work on soybean nodules has involved long-term treatments lasting at least 28 d, whilst the studies with lupin nodules suggested that changes in the extent of glycoprotein occlusion occurred over a period or hours or minutes (e.g. Iannetta et al. 1995 ). The original studies on the relationship between glycoprotein and oxygen diffusion in stressed soybean nodules ( James 1990) showed no clear changes in glycoprotein levels over the short term (minutes) or medium term (hours). However, this could reflect the rapid response to disturbance by the diffusion barrier in both control and treated soybean nodules prior to nodule fixation for microscopy ( Iannetta et al. 1993a ). Therefore, the occurrence of rapid changes in glycoprotein levels within soybean nodules has yet to be established.
Improvements in fixation techniques have occurred since the earlier work of James (1990), and the purpose of the present studies is to re-investigate the occurrence of medium-term changes in intercellular occlusions within soybean nodules. This involved comparing the structure of unstressed nodules with those removed from plants after a 3 h detopping treatment, known to affect oxygen diffusion resistance in soybean ( Minchin, Sheehy & Witty 1985; Denison et al. 1991 ).
MATERIALS AND METHODS
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.
RESULTS AND DISCUSSION
Western blots of glycoproteins
Although Western blot comparisons between MAC236 and MAC265 antigens have been reported for nodules of pea ( VandenBosch et al. 1989 ), lupin ( de Lorenzo et al. 1993 ) and Lotus ( James & Sprent 1999), such a comparison has not yet been reported for soybean. In Fig. 1, MAC265 antigen from soybean nodules shows a main band at ≈ 100 kDa (lane 2), which is the same as for pea nodules (lane 1). However, with both species, there are at least nine other bands covering a range from 80 to 200 kDa. Multiple banding with MAC265 was previously observed with pea nodule extracts ( VandenBosch et al. 1989 ; Rae et al. 1991 ) and with Lotus nodule extracts ( James & Sprent 1999) but not with lupin nodule extracts ( de Lorenzo et al. 1993 ; James et al. 1997 ). The MAC236 antigen from soybean nodules shows two much fainter bands at ≈ 130 and 210 kDa (lane 4), whilst that from pea nodules shows the same molecular weight as the main band for MAC265 (lane 3). The molecular weights for pea extracts are in agreement with those of VandenBosch et al. (1989) who reported values of 95 kDa for both antigens. These authors also reported that soybean nodule MAC236 had a higher molecular weight than that in pea. Variation in molecular weight between the two antigens has also been reported for lupin and Lotus nodules, although actual values were different from those reported here. In lupin nodules they were 240 and 135 kDa, for MAC236 and MAC265, respectively ( de Lorenzo et al. 1993 ), whereas in Lotus uliginosus nodules they were 170/210 (MAC236 and 155/170 (MAC265) ( James & Sprent 1999). Clearly, there is a need for further characterization of intercellular glycoproteins in legume nodules.
Glycoprotein levels in fresh nodules
To date, all immunolocalization of intercellular glycoproteins has involved sections of fixed nodule material and it is possible that the fixation process produces artefacts ( Van Cauwenberghe et al. 1993 ). To counter this, it is necessary to demonstrate the presence of glycoprotein antigens within fresh nodules. Tissue printing is a useful technique to localize antigens in fresh, unfixed, plant material ( Cassab & Varner 1987), and in this study ( Fig. 2a) shows that (a) the MAC236 antigen is abundant in nodules from detopped plants, (b) that it is primarily localized in the cortex internal to the scleroid layer, and (c) that some of the antigen is also present in the infected zone. These results were also found for the MAC265 antigen in detopped nodules and for both antigens with control nodules (data not shown).
However, a limitation of the tissue printing method is its lack of resolution, due to smearing of the antigen to be labelled, so that it was not possible to distinguish between antigen levels in the control and detopped nodules. This was resolved by the use of ELISAs which showed a significant increase in total MAC265 antigen level within soybean nodules following the 3 h detopping treatment ( Table 1). This suggests that intercellular glycoprotein deposition may increase following detopping, but such localization is beyond the resolution of the tissue printing method. Therefore, we examined immunostained cryosections of nodules to determine more precisely where the MAC236/MAC265 antigens are localized in fresh material. Even though the cortical tissue of the soybean nodules showed substantial disruption following cryosectioning ( Fig. 2b,d), it was possible to perform immunolabelling of this material to the extent of showing that the cortical cell wall area was a major location for the MAC236 antigen (compare Fig. 2b with Fig. 2d). However, due to the disruption of the tissue and the thickness of the sections (20 μm), it was not possible to determine whether the antigen labelling was in the intercellular spaces or the cell walls ( Fig. 2b). As the central infected region was more robust it remained intact, and, for the 3 h detopping treatment, clearly showed immunolabelling of intercellular glycoprotein ( Fig. 2c). MAC236 labelling of the infected zone was not observed with control nodules (data not shown), or in cortical sections incubated in blocking buffer alone ( Fig. 2d).
|ELISA (AMD g–1 fresh weight)||36||308 (21)||458 (46)**|
|Number of unoccluded ICS per section||24/22||82·0 (8·8)||20·7 (1·5)***|
|Area of unoccluded ICS (% of total section area)||24/22||0·58 (0·18)||0·16 (0·05)**|
|Area of labelled (occluded) ICS (% of total section area)||12||1·21 (0·25)||2·17 (0·37)**|
As well as providing new data with regard to glycoprotein within the infected zone, this study of fresh, unfixed soybean nodules has also provided information in support of previous studies of fixed embedded material ( VandenBosch et al. 1989 ; James et al. 1991 ). For example, those studies showed that, in soybean nodules, the glycoprotein was localized primarily within cortical intercellular spaces internal to the scleroid layer; the tissue prints and cryo-sections in the present study confirm this general pattern of localization in fresh material, and the cryosections confirm that the material can be intercellular. Therefore, it is reasonable to conclude that the localization of the MAC236/MAC265 glycoprotein shown in fixed material is generally representative of that in fresh nodules.
Immunolabelling of fixed nodules
Silver-enhanced, immunolabelled light micrographs of control soybean nodules show numerous open intercellular spaces (ICS) within the mid-cortex (using the terminology of Minchin 1997) with little or no labelling of glycoproteins ( Fig. 3a). However, following 3 h detopping, there appear to be far fewer unoccluded mid-cortical ICS, with labelling of the MAC236 antigen in both this region and the infected zone ( Fig. 3b). A higher-magnification light micrograph shows this labelling more clearly within occluded ICS in the mid-cortex region, including between the scleroid cells ( Fig. 3c). Labelling between the infected cells is towards the edge of the infected region, but appears to be largely random, and either occludes the ICS or occurs in the corners of otherwise open ICS ( Fig. 3d).
Transmission electron micrographs (TEMs) show the intercellular material within the infected region to be less dense and more lightly immunolabelled than that of the cortex (compare Fig. 4a,b with Fig. 4c). Material containing the MAC236/MAC265 antigen can also be found associated with electron-dense intercellular ‘plugs’ between infected cells ( Fig. 4d). This plate also shows unlabelled intercellular material which could correspond with other compounds which have been reported within cortical ICS (see Minchin 1997). TEMs of control nodules showed no MAC235/MAC265 antigen within the infected region but some labelling of ICS within the mid-cortex (mainly in the corners of ‘open’ spaces) and the scleroid layer (data not shown).
Morphometric analysis of cortical sections
The use of image analysis techniques allowed quantification of the number and area of unoccluded ICS within the mid and inner cortex regions (i.e. between the infected region and the scleroid cells). Using sections which were not immunolabelled showed that the 3 h detopping treatment produced a 70–75% reduction in both these parameters ( Table 1). With silver-enhanced immunolabelled sections it was possible to quantify the area of occluded ICS containing MAC236 antigen (e.g. Fig. 3c), which increased by a factor of 1·8 following detopping ( Table 1).
Improvements in fixation techniques have now allowed us to visualize and quantify an increased deposition of glycoprotein within ICS, especially within the mid-cortex region, following a 3 h detopping treatment. This glycoprotein could play a similar role in the regulation of O2 diffusion as that postulated for lupin nodules ( de Lorenzo et al. 1993 ; Iannetta et al. 1993a ). The purpose of glycoprotein (and other compounds, see Minchin 1997) within ICS is a matter for conjecture, but may well be related to the postulated hydrophobic nature of ICS ( Woolley 1983; Raven 1996), including those in the nodule cortex ( Webb & Sheehy 1991). Thus, the glycoprotein could provide a matrix for holding water within hydrophobic ICS. Given that changes in the water content of the ICS pathway are almost certainly involved in the operation of the variable oxygen diffusion barrier ( Minchin et al. 1985 ), it seems logical to propose that observed increases in glycoprotein-containing ICS occlusions are related to this process.
Nevertheless, in lupin nodules, O2-mediated intercellular occlusion production can occur within 15 min ( Iannetta et al. 1995 ), but there is still no evidence for such a rapid response with soybean nodules. Indeed, with soybean nodules, rapid responses by the oxygen diffusion barrier may be related to osmotic changes ( Purcell & Sinclair 1994; Serraj et al. 1995 ; Denison & Kinraide 1995) rather than glycoprotein-containing ICS occlusions.
The most surprising feature of the present data is the appearance of glycoprotein within the ICS of the infected region following a 3 h detopping treatment. The apparent random distribution and the low density of this material suggest that it is not directly involved in oxygen diffusion changes, which can be induced within a few minutes by detopping ( Minchin et al. 1985 ). It could represent a stress-induced ‘spill-over’ of glycoprotein from the cell walls; however, for reasons which are not clear, labelling of the antigen within cell walls of fixed nodules is uncommon (except for Sesbania rostrata stem nodules; James et al. 1996 ). In the present study, there may be labelling of cortical cell walls in the cryosections of fresh nodules, but this could be increased ICS labelling in these ‘thick’ sections.
Bergersen (1997) suggested, on theoretical grounds, that the extrusion of glycoproteins into the walls of infected cells could contribute to the regulation of free O2 entry into the cells. Also, Layzell (1998) argued that ‘a physical barrier to gas diffusion at the level of the infected cell membrane or cell wall would avoid the need for long-distance signal transduction’. The presence of glycoprotein within the infected region of stressed soybean nodules gives credence to these ideas and leads to speculation that glycoprotein deposition within cell walls may contribute towards a localized regulation of O2 diffusion into the infected cells. Such a regulation could produce very rapid ‘fine-tuning’ for O2 diffusion, whilst ICS occlusions and/or osmotic changes within the cortical region could provide a slower ‘coarse-tuning’.
We thank Dr N. J. Brewin for the gift of MAC236 and MAC265 antibodies, M. Gruber, H. Hodge and M. Kierans (Dundee University) for technical assistance, Dr I. Kill and Dr K. McElwee (Dundee University) for assistance in cryosectioning and I. Black (SCRI) for assistance with image analysis.
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