TEMPORAL RELATIONSHIPS BETWEEN NITROGENASE AND INTERCELLULAR GLYCOPROTEIN IN DEVELOPING WHITE LUPIN NODULES

The development of the N-2-fixing symbiosis between white lupin (Lupin us albus L.) cv. Multolupa and Bradyrhizobium strain ISLU16 was follow ed using the acetylene reduction assay (ARA), immunoblots of protein e xtracts, 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 macroscop ically visible nodule primordia had formed on roots by 8 d. The lack o f nitrogenase at these times was confirmed by a negative signal to imm unogold labelling with nitrogenase-specific antibodies. Ar 17 d three out of six plants had ARA, and nodules from these gave a positive sign al 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 antib ody. Western blots of nodule protein extracts using the monoclonal ant ibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprot ein which is considered to beinvolved in both rhizobial infection and the regulation ofnodule oxygen diffusion) gave a strong signal with no dules (fix+) from 20 d plants and with 17 d fix(-) plants. The signal with 1MAC236/MAC265 was substantially weaker with nodules from 17 d fi x- plants, and there was no signal apparent from nodules/nodulated roo ts from the 0, 8 and 12d harvests. However, further investigation usin g immunogold labelling revealed that not only were MAC236 and MAC265 e xpressed within cortical intercellular spaces in 20 d and 17 d fix(+)/ fix(-) nodules, but they were also strongly expressed in the developin g cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 1 2 d nodules. These data demonstrate that the glycoprotein recognized b y MAC'36 and MAC265 is present before the onset of nitrogenase express ion and function, but expression of the epitopes appears to be enhance d from the onset of N-2 futation. Nodules at all harvests were investi gated for the presence of infection threads, as the MAC236/MAC265-1 ec ognized glycoprotein is also a component of the infection thread matri x in nodules from other legumes. Infection threads were not seen in no dules from any of the harvests except for the 20 d nodules, and then o nly 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 me ristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majorit y of the IMAC236/MAC265 detected in lupin nodule extracts originated f rom glycoprolein within cortical intercellular spaces. (C) 1997 Annals of Botany Company.