Intercellular location of glycoprotein in soybean nodules: effect of altered rhizosphere oxygen concentration

Abstract

Abstract. A glycoprotein which occludes intercellular spaces in the inner cortex of legume nodules may be involved in controlling oxygen diffusion into rhizobial‐infected cells. Here we investigated this possibility by localizing the glycoprotein using monoclonal antibodies and immunogold labelling in nodulated roots of soybean cv. Clarke inoculated with Bradyrhizobium japonicum strain RCR3442 exposed to atmospheres with either 10, 21 (control) or 40% oxygen for 28d. Infected cells showed evidence of premature senescence when grown in above or below ambient pO2 particularly at 10% oxygen, although cortical cells appeared to be little altered by oxygen treatment. In the inner cortical cells, more glycoprotein was seen to be occluding intercellular spaces of those nodules subjected to 40% oxygen and less in those nodules exposed to 10% oxygen, when compared to controls. This observation, made at the light microscope level (using silver enhancement) was confirmed under the TEM using immunogold labelling. Therefore, it is suggested that intercellular space glycoprotein is one of the structural components of the diffusion resistance in the cortex of legume nodules.

Development of N2‐fixing nodules on the wetland legume Lotus uliginosus exposed to conditions of flooding

“…MAC236 and MAC265 were first shown to recognize epitopes on a glycoprotein component of the infection thread matrix and the infection droplet of pea and soybean nodules, as well as material within cortical intercellular spaces (VandenBosch et al, 1989 ;Rae et al, 1991Rae et al, , 1992James et al, 1991James et al, , 1992b. VandenBosch et al (1989) and Rae et al Fig.…”

“…The plants were grown in a glasshouse under natural light supplemented with artificial light from 400 W mercury vapour bulbs in order to give a photoperiod of 16 hr light : 8 hr dark, and a light\dark temperature of 20\15mC. Plants sown in the waterfilled tank were kept continuously flooded up to 1 cm above the hypocotyl, the water within the tank being supplemented weekly with an N-free nutrient solution (James et al, 1991) that was also given to the non-flooded plants. Most plants were harvested at 60 d after sowing for dry weight determination, acetylene reduction assays (ARAs) and microscopy.…”

“…The occurrence of infection threads within roots was examined using the tissue clearing\methylene blue-staining method (Vasse & Truchet, 1984). Nodules of all ages were fixed in glutaraldehyde and embedded in LR White resin for light microscopy and subjected to immunogold labelling according to James et al (1991). Nodules were also prepared for standard transmission electron microscopy (TEM) by post-fixing with osmium tetroxide before dehydration and embedding (James et al, 1992b).…”

“…Undetached nodules on intact plants were assessed for the presence of active nitrogenase using the ' closed ' ARA according to James et al (1991). The occurrence of infection threads within roots was examined using the tissue clearing\methylene blue-staining method (Vasse & Truchet, 1984).…”

“…Immunogold labelling for light microscopy (with silver enhancement) and TEM using the monoclonal antibodies MAC236 and MAC265 (kind gifts from Dr N. J. Brewin, John Innes Centre, Norwich, UK) was performed according to VandenBosch et al (1989) and James et al (1991), and that using the antibody raised against nitrogenase was performed according to James et al (1996). In the case of MAC236 and MAC265, goat anti-rat antibodies conjugated to 10 nm gold particles were used for TEM, and goat anti-rat antibodies conjugated to 5 nm gold particles, combined with the Intense M silver enhancement kit, were used for optical microscopy.…”