Leghemoglobin in Lupin Plants

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus aIbus cv Multolupa inoculated with Bradyrhizoblum sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate. The hemoglobins from legume nodules (leghemoglobins, LHbs) are monomeric proteins that function by providing an optimum concentration of 02 to bacteroids in order to maintain nitrogenase and respiratory activities. They have unique properties which enable them to facilitate the flux of oxygen to the Rhizobium endophyte at an extremely low free-oxygen concentration. Without leghemoglobin in the peribacteroid space, oxygen would be deficient through much of the space, the 02 flux could not be maintained, and the bacteroid respiration would be greatly reduced. Rates of nitrogen fixation in nodules from various species of legumes are believed to be limited by the resistance to the diffusion of oxygen (30). Leghemoglobin localization has been a controversial subject (5, 28). Robertson et al. (25) studied this by immunocytochemistry in peas and found LHb in the cytosol and, unexpectedly, in nuclei; they found no LHb in the peribacteroid space. Therefore, confirmation of the sites of localization seemed important with regard to other legumes. Moreover, Bisseling et al. (6) found that the addition of combined nitrogen decreased the amount of leghemoglobin, there being a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. Recently, other authors (3) gave no importance to a negative effect of nitrates directly on nitrogenase activity, in favor of barriers to ' Supported by a grant from the Comision Asesora de Investigaci6n Cientifica y Tecnica of Spain (Project 797). gas diffusion in nodules. Witty et al. (30) also concluded that rates of nitrogen fixation in nodules from various legumes are limited by the resistance to the diffusion of oxygen. For our study, we used lupin plants (Lupinus albus L. cv Multolupa), a crop of importance in Spain as an alternative crop to soybean. Nitrogen compounds formed in the lupin nodules are exported as amides. The central region of the nodule consists entirely of bacteroid-infected cells. The nitrogen-fixing bacteroids are encircled (one or several bacteroids) by the peribacteroid membrane and the peribacteroid spaces are relatively large. Our main objective was to localize LHb in nodule cells of lupin. We also studied the effect ofcombined nitrogen on the immunocytochemical localization and amount of LHb. MATERIALS AND METHODS