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Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O2 and short‐term N2 deprivation (1h in Ar:O2). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H2 evolution in a gas exchange system. Respiration was simultaneously recorded as CO2 evolution. Dark‐treated plants had lower initial nitrogenase activity in N2:O2 (68% of controls), which declined further during a 1‐h period in the assay system in N2:O2 at 21 and 17% O2, but not at 13% O2. When dark‐treated plants were deprived of N2 at 21 and 17% O2 nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O2, and only a smaller and temporary decline in control plants at 21% O2. When dark‐treated plants were kept at 21% O2 during 45 min prior to N2 deprivation at 17% O2 the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N2:O2 at 21% O2 and during N2 deprivation was caused by O2, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N2 deprivation, suggesting decreased assimilation of NH4+. Contents of ATP and ADP in nodules were not affected by short‐term N2 deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O2. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO2 and during cessation of NH4+ production. [ABSTRACT FROM AUTHOR]

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus intana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro (Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that. in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the. nitrogenase proteins were the only abundant proteins tint clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity. Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation. [ABSTRACT FROM AUTHOR]