Temporal relationships between nitrogenase and intercellular glycoprotein in developing white lupin nodules

The development of the N₂-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 20d 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 17d fix plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12d 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 N₂ 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.