N-2 fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants

Common beans (Phaseolos vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N-2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N(2)ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O-2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N(2)ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N(2)ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O-2 in the infected region.