Nitrogenase Activity in Alnus incana Root Nodules. Responses to O2 and Short-Term N2Deprivation

Abstract

O 2 and host-microsymbiont interactions are key factors affecting the physiology of N 2 -fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O 2 concentration, and short-term N 2 deprivation, intact nodulated roots were exposed to various O 2 pressures (pO 2 ) and Ar:O 2 in a continuous flow-through system. Nitrogenase activity (H 2 production) occurred at a maximal rate at 20% O 2 . Exposure to shortterm N 2 deprivation in Ar:O 2 carried out at either 17%, 21%, or 25% O 2 caused a decline in the nitrogenase activity at 21% and 25% O 2 by 12% and 25%, respectively. At 21% O 2 , nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N 2 fixation. Respiration (net CO 2 evolution) decreased in response to the N 2 deprivation at all pO 2 values and did not recover during the time in Ar:O 2 . Increasing the pO 2 from 21% to 25% and decreasing the pO 2 from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O 2 limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O 2 inhibition of nitrogenase at 25% O 2 . These results are consistent with a fixed O 2 diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O 2 and N 2 deprivation.

Primary metabolism in N2-fixing Alnus incana?Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

“…The plants were grown as described earlier (Lundquist 2000) and the nodules were used 8 weeks after inoculation. The nitrogen-fixing nodules produced on the roots were used for perfusion experiments, as well as for metabolite extracts.…”

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

“…By making use of a unique strain of Frankia lacking hydrogenase uptake activity in the present study, it was possible to perform continuous measurements of nitrogenase activity in N 2 :O 2 thus without disturbing N 2 fixation. Alnus nodules lack a variable diffusion barrier to O 2 (Lundquist, 2000), in contrast to crop legumes, and the EAC is unaffected by O 2 concentration in the range investigated in the present study (Lundquist, 2000). This makes short-term assays of apparent nitrogenase activity (ANA) more stable and representative of preassay activity as demonstrated for plants exposed to prolonged darkness (Lundquist et al, 2003) and probably proportional to N 2 fixation.…”

“…In other hosts such as Casuarina and Myrica, the host part of the nodule appear to contribute in reducing O 2 concentration within the nodule (Silvester et al, 1990;Winship and Silvester, 1989). In contrast to the legume root nodules, there does not seem to be a short-term variable diffusion barrier to O 2 in Alnus nodules (Lundquist, 2000). Alnus nodules appear to have an overall aerobic metabolism as indicated by a high ATP/ADP ratio, in contrast to lower ratios in the micro-aerobic legume nodules (Lundquist et al, 2003).…”

“…Plant material and growth conditions have been described earlier (Lundquist, 2000). In brief, rooted cuttings of a clone of grey alder (Alnus incana (L.) Moench) were inoculated with a crushed nodule inoculum lacking hydrogenase uptake activity (the local source of Frankia; HussDanell, 1991;Sellstedt et al, 1986), and planted into pots containing granite-rock gravel.…”

“…By applying short-term gas-exchange assays in two different approaches it was possible to obtain results for non-stressed plants. Complete abolishment of N 2 fixation is known to cause declines in nitrogenase activity and respiration whether it is during an acetylene reduction assay (ARA) or during assays of nitrogenase-dependent H 2 production in an atmosphere of Ar:O 2 (Lundquist, 2000;Lundquist et al, 2003;Rosendahl and Huss-Danell, 1988;Tjepkema et al, 1988). By making use of a unique strain of Frankia lacking hydrogenase uptake activity in the present study, it was possible to perform continuous measurements of nitrogenase activity in N 2 :O 2 thus without disturbing N 2 fixation.…”