Proteolytic Activity in Soybean Root Nodules 1 ACTIVITY IN HOST CELL CYTOSOL AND BACTEROIDS THROUGHOUT PHYSIOLOGICAL DEVELOPMENT AND SENESCENCE

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merril cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis. Nitrogenase activity profiles of nodules have been followed throughout the developmental cycle of soybean plants by a number of investigators (9, 10, 13, 20). Total nitrogenase activity per plant, measured by the acetylene reduction assay, generally peaks at full flowering or as pod formation begins, and then declines as seeds develop (13, 20). However, the extent of this decline appears to vary considerably and is dependent upon such factors as plant cultivar (13), row spacing (25), environmental conditions such as temperature and photoperiod, and whether plants are fieldor chamber-grown. In some cases, nitrogenase activity exhibited an initial decline during pod formation, but then was maintained at a significant level throughout the period of seed fill (9, 10, 17). Some investigators have used the initial decline in nitrogenase activity as an indication that nodule senescence has begun (12), even though significant activity may remain for quite some time. Others (19) have defined nodule senescence as the period of rapid nodule decay which occurred at physiological maturity (leaf yellowing). Additional problems arise when investigators fail to give any indication of the developmental stage of the pfant when monitoring nitrogenase activity. Since the number of days from planting to harvest maturity depends upon plant cultivar and growth conditions, in many cases it is difficult to relate nodule senescence, as defined by a decline in nitrogenase activity, with a particular stage of plant development. 'Supported in part by United States Department of Energy/Science and Education Administration-CRGO Grant No. 509-0410-9-0233 and the Nebraska Soybean Development, Utilization and Marketing Board, Contract No. 380-82-12. Published as Paper No. 6982, Journal Series, Nebraska Agricultural Experiment Station. 2To whom all communications should be addressed. Declines in total soluble protein (15) and total leghemoglobin (12) contents have been observed in senescing soybean nodules, although these losses generally occurred later than the initial decline in nitrogenase activity. Similar declines in soluble protein and leghemoglobin contents occurred in senescing alfalfa (Medicago sativa L.) nodules following foliage harvest (24) which were correlated with an increase in proteolytic activity. It has been postulated (2) that autolytic processes in the bacteroids and host legume cells were probably responsible for the senescence of legume nodules, and Truchet and Coulomb (21) suggest that 'phytolysosomes' i.e. vacuoles, may be involved in the cellular disorganization that occurs at senescence in pea (Pisum sativum L.) nodules. Previous work in this laboratory has identified and partially characterized the proteolytic enzymes of soybean nodule cytosol (14). This report describes the peptidases found in bacteroid extracts, monitors these activities in both host cells and bacteroids throughout the developmental cycle of soybean plants, and relates these activities to changes in nitrogenase activity that occur throughout the period of pod formation, seed development, and maturation. MATERIALS AND METHODS Seed Inoculation and Plant Growth. The methods used to prepare inoculum, to infect soybean seeds (Glycine max L. Merrill cv Woodworth) and to grow soybean plants have been reported previously (14), except that Rhizobium japonicum (strain 61A89) was used and each 15-cm pot contained only one plant. After plants had nodulated (at about the time when the first trifoliate leaf was fully expanded), they were repotted in sterile Arcil lite (IMC Chemical Corp.), an inert root support. They were grown at 26 ± 2°C in an environmentally controlled chamber on a 16-h photoperiod until flowering commenced, and thereafter on a 14-h photoperiod. Light intensity averaged 700 ,uE Plants were watered twice daily and given nitrogen-free Hoagland solution twice weekly. Acetylene Reduction. Nitrogen fixation was measured on intact plants using the acetylene reduction assay (7) as reported previously (16). Nodule Harvest. Root nodules were harvested throughout the developmental cycle of the soybean plants at 1to 2-week intervals. The first nodules were collected during early vegetative growth, as soon as nodules were large enough to be picked from the root, and the last nodules were collected when the plants had reached harvest maturity. At each harvest date, the physiological development of the plants was classified according to the descriptions suggested by Fehr et al. (5) for developmental stages of soybean growth. As many nodules as possible were harvested from the plants on the day following acetylene reduction assays. All nodules from a single plant were pooled, weighed, frozen, and stored at -80°C until used. A l-g aliquot of nodules was counted