Oxygen-Induced Membrane Depolarizations in Legume Root Nodules (Possible Evidence for an Osmoelectrical Mechanism Controlling Nodule Gas Permeability).

Various stresses trigger rapid and reversible decreases in the O2 permeability (PO) of legume root nodules. Several possible mechanisms have been proposed, but no supporting data have previously been presented that meet the requirements for both rapidity and reversibility. Stomatal regulation of gas permeability in leaves involves electrically driven fluxes of inorganic osmoticants, so we investigated the possibility of a somewhat similar mechanism in nodules. We used microelectrodes to monitor membrane potential in intact, attached nodules of Glycine max, Medicago sativa, Lotus corniculatus, and Trifolium repens while controlling external O2 concentration and, in the case of G. max, measuring PO with a nodule oximeter. A 1- to 2-min exposure to 100 kPa O2 was found to induce rapid and reversible membrane depolarizations in nodules of each species. This depolarization (which, to our knowledge, is unique to nodules) is accompanied by reversible decreases in PO in G. max nodules. An osmoelectrical mechanism for control of nodule gas permeability, consistent with these data, is presented.

Nitrate Effects on Nodule Oxygen Permeability and Leghemoglobin (Nodule Oximetry and Computer Modeling).

Two current hypotheses to explain nitrate inhibition of nodule function both involve decreased O2 supply for respiration in support of N2 fixation. This decrease could result from either (a) decreased O2 permeability (PO) of the nodule cortex, or (b) conversion of leghemoglobin (Lb) to an inactive, nitrosyl form. These hypotheses were tested using alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus) plants grown in growth pouches under controlled conditions. Nodulated roots were exposed to 10 mM KNO3 or KCI. Fractional oxygenation of Lb under air (FOLair), relative concentration of functional Lb, apparent PO, and O2-saturated central zone respiration rate were all monitored by nodule oximetry. Apparent PO and FOLair in nitrate-treated nodules decreased to <50% of values for KCI controls within 24 h, but there was no decrease in functional Lb concentration during the first 72 h. In nitrate-treated alfalfa, but not in birdsfoot trefoil, FOLair, apparent PO, and O2-saturated central zone respiration rate decreased during each light period and recovered somewhat during the subsequent dark period. This species difference could be explained by greater reliance on photoreduction of nitrate in alfalfa than in birdsfoot trefoil. Computer simulations extended the experimental results, showing that previously reported decreases in apparent PO of Glycine max nodules with nitrate exposure cannot be explained by hypothetical decreases in the concentration or O2 affinity of Lb.

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules.

Various forms of stress result in decreased O(2) permeability or decreased capacity to consume O(2) in legume root nodules. These changes alter the nodule interior O(2) concentration (O(i)). To determine the relationship between O(i) and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O(i) by varying external pO(2) while monitoring internal H(2) concentration (H(i)) with microelectrodes. O(i) was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O(i), H(i) approached a new steady state, with a time constant averaging 23 s. The rate of H(2) production by nitrogenase was calculated as the product of H(i), nodule surface area, and nodule H(2) permeability. H(2) permeability was estimated from O(2) permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O(i) was nearly optimal for nitrogenase activity (H(2) production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO(2) (40-100 kPa) reduced H(i) to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO(2). The mechanism of nitrogenase inhibition may differ between brief and prolonged O(2) exposures.

Mathematical modeling of oxygen diffusion and respiration in legume root nodules.

The O(2) permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O(2) and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O(2) or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O(2) permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O(2) method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O(2) and uniform conditions in the nodule interior) are met.

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil.

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H(2) evolution in Ar:O(2) (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO(2) production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O(2) permeability decreased 50% within 5 hours in each species. A similar decrease in the O(2)-saturated respiration rate (V(max)) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V(max) in each species. V(max) may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O(2) inactivation of nitrogenase.

Measurement of legume nodule respiration and o(2) permeability by noninvasive spectrophotometry of leghemoglobin.

Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V(max). The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.

Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans.

The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit-nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit-nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans.

Analysis of acetylene reduction rates of soybean nodules at low acetylene concentrations.

It has been previously proposed that acetylene reduction data at subsaturating acetylene concentrations could be interpreted by use of the Michaelis-Menten equation, based on the acetylene concentration external to the nodules. One difficulty of this view is that the assumption that the system is not diffusion limited is violated when studying intact nodules. The presence of a gas diffusion barrier in the nodule cortex leads to an alternate expression for the gas exchange rates at subsaturating gas concentrations. A theoretical comparison of the ;apparent' Michaelis-Menten model and diffusion model illustrated the difficulties observed in the former model of overestimating the Michaelis-Menten coefficient and yielding a correlation between the Michaelis-Menten coefficient and the maximum rate. On the other hand, use of a diffusion model resulted in (a) estimates of the Michaelis-Menten coefficient consistent with enzyme studies, (b) stability of the estimates of the Michaelis-Menten coefficient independent of treatment, and (c) a sensitivity of the diffusion barrier conductance to plant drought stress. It was concluded that all studies of nodule gas exchange need to consider possible effects caused by the presence of a diffusion barrier.