J Plant Physiol. Vol. 144. pp. 633-640 (1994) Introduction Protective Enzymes against Active Oxygen Species during Nitrate-Induced Senescence of Lupinus a/bus Nodules* CRISTINA A. DE LORENZO, M. MERCEDES FERNANDEZ-PASCUAL, and M. ROSARIO DE FELIPE Departamento de Fisiologfa y Bioqufmica Vegetal, Centro de Ciencias Medioambientales, CSIC, Serrano 115 Dpdo., 28006 Madrid (Spain) Received February 7,1994 . Accepted June 6,1994 Summary Nitrate application to nodulated legumes inhibits nitrogenase activity and causes the premature senes- cence of nodules. In this work the activity of antioxidant enzymes and the production of toxic oxygen species during nitrate-induced senescence of Lupinus albus L. cv. Multolupa nodules was investigated. Four-six days of nitrate treatment induced a decrease in the activity of the main enzymes that scavenge H 2 0 2 in the nodule cytosol, catalase and ascorbate peroxidase. Total peroxidase activity increases in re- sponse to nitrate treatment, but H 20 2 eventually builds up in nodule cytosol. It is suggested that this H 2 0 2 causes the first damage to leghemoglobin apoprotein, which results in a decreased in situ immuno- labelling of this hemoprotein. Degradation of Lb continues with the rupture of the heme group after 8 days of nitrate treatment. This may result in the release of catalytic iron, which in turn may promote the formation of hydroxyl radicals from the precursors H 2 0 2 and O 2 , or from H 2 0 2 through a Fenton- type reaction. Key words: Free radicals, leghemoglobin, Lupinus albus, nitrate, nodules. Abbreviations: AOS = active oxygen species; ASC = ascorbate; BSA = bovine serum albumin; DTT = dithiothreitol; Cu,Zn-SOD = cupro-zinc-containing superoxide dismutase; EDTA = ethylenediamine tetracetic acid; Lb = leghemoglobin; Lb 2 +, Lb 3 + = ferrous and ferric leghemoglobin; Lb.0 2 = oxyleghe- moglobin; Mn-SOD = manganese-containing superoxide dismutase; NBT = nitro blue tetrazolium; o-di- anisidine = 3,3' -dimetoxybenzidine; PVPP = polyvinylpolypirrolidone; Rm = relative mobility; SOD = superoxide dismutase; Tricine = N-(tris[hydroxymethyl]methyl)glycine. L. cv. Multolupa) nodules subjected to several stresses, among these, exposure to KN0 3 for several days. The oxygen relations of legume nodules are extremely im- portant in maintaining nodule functions; therefore, in the past few years the central role of oxygen in nodule metabo- lism has been acknowledged. In respect to molecular oxygen (02), nodules possess a regulatory barrier to O 2 diffusion (Layzell and Hunt, 1991). The barrier has recently been identified Gannetta el al., 1993; de Lorenzo et al., 1993) and characterized (Minchin et al., 1992) in lupine (Lupinus albus The inhibitory effect of nitrate on nitrogenase has been a matter of research for many decades. According to Minchin et al. (1989), the latter stages of the nitrate-induced decline would permit the access of nitrate to the infected zone, and its possible reduction to nitrite. Although the role of nitrite is not still clear, these latter stages of nitrate treatment lead to irreversible nodule senescence. In this sense, it is well known that senescence processes have been traditionally associated with an increased produc- tion of toxic active oxygen species (AOS) and lowered levels of the protective enzymes catalase (EC 1.11.1.6), peroxidase * Dedicated to Prof. Dr. H. Lichtenthaler on the occasion of his 60th birthday. © 1994 by Gustav Fischer Verlag, Stuttgart